THEREOF

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. 119(e) of U.S. provisional application number 63/248,968 filed on September 27, 2021 which is incorporated by reference herein in its entirety.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (V029170023WO00-SEQ-CEW; Size: 225,278 bytes; and Date of Creation: September 26, 2022) is herein incorporated by reference in its entirety.

BACKGROUND

Clustered regulatory Interspaced Short Palindromic Repeats (CRISPR)/Cas systems a provide a platform for targeted gene editing in cells. Despite the versatility of the systems and associated tools for use, there are a number of limitations in these tools for the specific introduction of targeted modifications into the cell genome, for example, for modifying the coding sequence of a gene associated with a disease or disorder.

SUMMARY

The disclosure is directed, in part, to fusion polypeptides comprising a Cpf 1 domain that is catalytically inactive (lacks nuclease activity) and an endonuclease domain (e.g., from a restriction endonuclease, such as FokI) that function in directing single stranded DNA cleavage (i.e., nickase activity) to a target site in the genome of a cell.

Accordingly, in one aspect, the disclosure is directed to a fusion polypeptide comprising a Cpf 1 domain that lacks nuclease activity, and an endonuclease domain.

In some embodiments, the endonuclease domain comprises a first DNA-cleavage domain of a restriction endonuclease, wherein the first DNA-cleavage domain is capable of forming a dimer with a second DNA-cleavage domain of a restriction endonuclease. In some embodiments, the endonuclease domain comprises a first DNA-cleavage domain of a restriction endonuclease and a second DNA-cleavage domain of a restriction endonuclease, wherein the first DNA-cleavage domain and second DNA-cleavage domain are capable of forming a dimer with one another. In some embodiments, the dimer of the first and second DNA-cleavage domain is capable of producing a single strand break in DNA.

In some embodiments, the restriction endonuclease is a type IIS restriction endonuclease or portion thereof. In some embodiments, the endonuclease domain comprises FokI or a portion thereof. In some embodiments, the first and/or second DNA-cleavage domain is a DNA cleavage domain of FokI or derived therefrom. In some embodiments, the endonuclease domain does not comprise the DNA binding domain of FokI and/or is not capable of forming and/or maintaining a complex with DNA in the absence of an accompanying Cpfl domain. In some embodiments, the first DNA-cleavage domain or the second DNA-cleavage domain comprises one or more modifications relative to a corresponding wildtype sequence. In some embodiments, the one or more modifications alter activity of the endonuclease domain such that the endonuclease domain does not produce double strand breaks in DNA. In some embodiments, the one or more modifications decrease or eliminate endonuclease activity of the endonuclease domain. In some embodiments, the endonuclease domain comprises an amino acid sequence of any of SEQ ID NOs: 13 or 14, or a sequence with at least 80, 85, 90, 95, or 99% identity to any thereof.

In some embodiments, the Cpfl domain comprises an amino acid sequence of a Cpfl protein from Prevotella spp., Francisella spp., Acidaminococcus sp. (AsCpfl), Lachnospiraceae bacterium (LpCpfl), Eubacterium rectale, or an engineered Cpfl. In some embodiments, the Cpfl domain comprises one or more amino acid modifications relative to a corresponding wildtype Cpfl amino acid sequence. In some embodiments, the one or more modifications comprise one or more amino acid substitutions in the Cpfl protein relative to the wildtype sequence. In some embodiments, the Cpfl domain comprises a substitution at: one, two, three, or each of amino acids corresponding to positions 174, 542, 548, or 552 of the Acidaminococcus sp. Cpfl amino acid sequence. In some embodiments, the Cpfl domain comprises a substitution at: one, two, three, or each of amino acids corresponding to positions 169, 529, 535, or 538 of the MAD7™ Cpfl amino acid sequence provided by SEQ ID NO: 1. In some embodiments, the one or more substitutions comprise an arginine at the position corresponding to position 174, an arginine at the position corresponding to position 542, a valine at the position corresponding to position 548, and/or an arginine at the position corresponding to position 552 of the Acidaminococcus sp. Cpfl amino acid sequence provided by SEQ ID NO: 4.

In some embodiments, the one or more substitutions comprise an arginine at the position corresponding to position 169, an arginine at the position corresponding to position 529, a valine at the position corresponding to position 535, and/or an arginine at the position corresponding to position 538 of the MAD7™ Cpfl amino acid sequence provided by SEQ ID NO: 1.

In some embodiments, the fusion polypeptide further comprises c) a genomic modification domain. In some embodiments, the genomic modification domain comprises a base editor. In some embodiments, the base editor is a cytosine base editor (CBE) or an adenine base editor (ABE). In some embodiments, the base editor comprises a cytidine deaminase or an adenine deaminase. In some embodiments, the base editor comprises both a cytidine deaminase and an adenine deaminase. In some embodiments, the genomic modification domain comprises an epigenetic modifier. In some embodiments, the epigenetic modifier comprises a DNA methyltransferase, a DNA methylase, a histone acetyltransferase, a histone deacetylase, a histone methyltransferase, a histone methylase, or a functional portion or combination of any thereof. In some embodiments, the genomic modification domain comprises an amino acid sequence of SEQ ID NO: 15, or a sequence with at least 80, 85, 90, 95, or 99% identity to any thereof.

In some embodiments, the Cpfl domain is N-terminal of the endonuclease domain. In some embodiments, the endonuclease domain is N-terminal of the Cpfl domain. In some embodiments, the genomic modification domain is N-terminal of the Cpfl domain. In some embodiments, the genomic modification domain is N-terminal of the endonuclease domain. In some embodiments, the fusion comprises from N-terminus to C-terminus: the Cpfl domain, the endonuclease domain, and the genomic modification domain. In some embodiments, the fusion comprises from N-terminus to C-terminus: the Cpfl domain, the genomic modification domain, and the endonuclease domain. In some embodiments, the fusion comprises from N-terminus to C-terminus: the endonuclease domain, the Cpfl domain, and the genomic modification domain. In some embodiments, the fusion comprises from N-terminus to C-terminus: the endonuclease domain, the genomic modification domain, and the Cpfl domain. In some embodiments, the fusion comprises from N-terminus to C- terminus: the genomic modification domain, the Cpfl domain, and the endonuclease domain. In some embodiments, the fusion comprises from N-terminus to C-terminus: the genomic modification domain, the endonuclease domain, and the Cpfl domain. In some embodiments, the fusion polypeptide further comprises one or more linker domains. In some embodiments, the linker is an XTEN linker.

In another aspect, the disclosure is directed to a nucleic acid comprising a nucleotide sequence encoding a fusion polypeptide described herein.

In another aspect, the disclosure is directed to a vector comprising a nucleic acid described herein.

In another aspect, the disclosure is directed to a cell comprising a fusion polypeptide, the nucleic acid, or vector described herein.

In another aspect, the disclosure is directed to a system comprising: a fusion polypeptide described herein; and a first gRNA comprising a targeting domain complementary to a first target sequence in the genome of a cell, wherein the fusion polypeptide is capable of forming and/or maintaining a ribonucleoprotein (RNP) complex with the first gRNA and the RNP complex is capable of binding the target sequence in the genome of a cell. In some embodiments, the system further comprises a second gRNA comprising a targeting domain complementary to a second target sequence in the genome of the cell, wherein the first and second target sequences are not the same. In some embodiments, the system further comprises a second fusion polypeptide comprising a) a Cpf 1 domain that lacks nuclease activity, and b) a second endonuclease domain capable of forming a dimer with the first endonuclease domain.

In another aspect, the disclosure is directed to a ribonucleoprotein (RNP) complex comprising: a fusion polypeptide described herein; and a gRNA comprising a targeting domain complementary to a target sequence in the genome of a cell, wherein RNP complex is capable of binding the target sequence in the genome of a cell.

In another aspect, the disclosure is directed to a method comprising: i) contacting a cell with a fusion polypeptide or nucleic acid described herein; and ii) contacting the cell with a first gRNA comprising a targeting domain complementary to a first target sequence in the genome of a cell. In some embodiments, i) and ii) occur simultaneously or in close temporal proximity. In some embodiments, the method further comprises: iii) contacting the cell with a second gRNA (or nucleic acid encoding the same) comprising a targeting domain complementary to a second target sequence in the genome of a cell. In some embodiments, the method further comprises contacting the cell with a second fusion protein or nucleic acid described herein.

In another aspect, the disclosure is directed to a method, comprising: i) contacting a cell with a first fusion polypeptide described herein and a first gRNA comprising a targeting domain complementary to a first target sequence in the genome of a cell; and ii) contacting the cell with a second fusion polypeptide described herein and a second gRNA comprising a targeting domain complementary to a second target sequence in the genome of a cell, wherein the first target sequence and the second target sequence are not the same and the first fusion polypeptide and second fusion polypeptide are not the same.

In some embodiments, the first target sequence and the second target sequence are on different chromosomes of the genome of the cell. In some embodiments, the first target sequence and the second target sequence are on the same chromosome in the genome of the cell. In some embodiments, the first target sequence and the second target sequence are on the same DNA strand of the chromosome. In some embodiments, the first target sequence and the second target sequence are on different DNA strands of the chromosome. In some embodiments, the first target sequence and the second target sequence are separated by 10- 10,000 nucleotides.

In some embodiments, the cell is a hematopoietic cell. In some embodiments, the cell is a hematopoietic stem cell. In some embodiments, the cell is a hematopoietic progenitor cell. In some embodiments, the cell is an immune effector cell. In some embodiments, the cell is a lymphocyte. In some embodiments, the cell is a T-lymphocyte.

In another aspect, the disclosure is directed to an engineered cell, or descendant thereof, produced by a method described herein.

In another aspect, the disclosure is directed to a cell population, comprising an engineered cell described herein.

In another aspect, the disclosure is directed to a chimeric polypeptide that lacks nuclease activity, comprising: a first portion comprising an amino acid sequence of a first Cpf 1 protein, and a second portion comprising an amino acid sequence of a second Cpfl protein, wherein the first Cpfl protein and second Cpfl protein are not the same. In some embodiments, the first Cpfl protein is derived from a Cpfl from Prevotella spp. or Francisella spp., Acidaminococcus sp. (AsCpfl), Lachnospiraceae bacterium (LpCpfl), or Eubacterium rectale, or MAD7™ as provided by Inscripta. In some embodiments, the second Cpfl protein is derived from a Cpfl from Prevotella spp. or Francisella spp., Acidaminococcus sp. (AsCpfl), Lachnospiraceae bacterium (LpCpfl), or Eubacterium rectale, or MAD7™ as provided by Inscripta. In some embodiments, the first Cpfl protein comprises an Acidaminococcus sp. Cpfl (AsCpfl) or portion thereof. In some embodiments, the second Cpfl protein comprises MAD7™ or a portion thereof. In some embodiments, the first Cpfl protein and/or second Cpfl protein comprise one or more modifications relative to the wildtype sequence of the first Cpfl protein and/or second Cpfl protein. In some embodiments, the one or more modifications comprise one or more amino acid substitutions in the first Cpfl protein relative to the wildtype sequence of the first Cpfl protein.

In some embodiments, the amino acid sequence comprising the first Cpfl protein is at least 100 amino acids in length, or 100-1300 amino acids in length. In some embodiments, the amino acid sequence comprising the second Cpfl protein is at least 100 amino acids in length, or 100-1300 amino acids in length. In some embodiments, the chimeric polypeptide further comprises a linker between the first portion and second portion. In some embodiments, the chimeric polypeptide is at least 800 amino acids in length, or 800-1500 amino acids in length.

In some embodiments, the amino acid sequence of the first Cpfl protein comprises any of SEQ ID NOs: 1-9 or a sequence with at least 80, 85, 90, 95, or 99% identity to any thereof. In some embodiments, the amino acid sequence of the second Cpfl protein comprises any of SEQ ID NOs: 1-9 or a sequence with at least 80, 85, 90, 95, or 99% identity to any thereof. In some embodiments, the chimeric polypeptide comprises an amino acid sequence of any of SEQ ID NOs: 24-31 or a sequence with at least 80, 85, 90, 95, or 99% identity to any thereof.

The summary above is meant to illustrate, in a non-limiting manner, some of the embodiments, advantages, features, and uses of the technology disclosed herein. Other embodiments, advantages, features, and uses of the technology disclosed herein will be apparent from the Detailed Description, the Drawings, the Examples, and the Claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic of an exemplary plasmid vector encoding an enhanced Cpfl nuclease (enCpfl, enAsCpfl-(RVR)). The vector encodes a gRNA scaffold under the control of the U6 promoter. The enhanced Cpfl is a Cpfl nuclease from Acidaminococcus sp. BV3L6 containing the E174R/S542R/K548V/N552R mutations (referred to as “enAsCpfl-(RVR)”) under the control of the chicken beta- actin promoter and cytomegalovirus (CMV) enhancer sequence.

FIG. 2 shows a schematic of an exemplary plasmid vector encoding a fusion of a base editor and an enhanced Cpfl nuclease. The vector encodes a gRNA scaffold under the control of the U6 promoter. The base editor-Cpfl fusion is a fusion of the exemplary base editor APOBEC-1 fused to the N-terminus of the enhanced Cpfl nuclease from Acidaminococcus sp. BV3L6 containing the E174R/S542R/K548V/N552R mutations under the control of the chicken beta-actin promoter and CMV enhancer sequence.

FIG. 3 shows a schematic of an exemplary plasmid vector encoding the MAD7™ nuclease. The vector encodes a gRNA scaffold under the control of the U6 promoter. The gene encoding MAD7™ (Inscripta) is under the control of the chicken beta-actin promoter and CMV enhancer sequence.

FIG. 4 shows a schematic of an exemplary plasmid vector encoding an enhanced nuclease based on the MAD7™ nuclease. The vector encodes a gRNA scaffold under the control of the U6 promoter. The enhanced nuclease based on the MAD7™ nuclease contains the mutations K169R, D529F, K535V, and N538R under the control of the chicken beta-actin promoter and CMV enhancer sequence.

FIG. 5 shows a schematic of an exemplary plasmid vector encoding a fusion polypeptide comprising a Cpfl domain lacking nuclease activity (dCpfl) and two FokI nuclease domains (Fokl domain I and Fokl domain II The vector encodes a gRNA scaffold under the control of the U6 promoter. The fusion polypeptide is a fusion of the two Fokl nuclease domains to the C-terminus of a nuclease-dead Cpfl enzyme from Acidaminococcus sp. BV3E6 containing a D908A mutation under control of the chicken beta actin promoter and CMV enhancer sequence. The Fokl domains are separated from each other with a polypeptide linker and from dCpf 1 with an XTEN linker.

FIG. 6 shows a schematic of an exemplary plasmid vector encoding a fusion polypeptide comprising a Cpfl domain lacking nuclease activity (dCpfl) and two Fokl nuclease domains (Fokl domain I and Fokl domain II). The vector encodes a gRNA scaffold under the control of the U6 promoter. The fusion polypeptide is a fusion of the two Fokl nuclease domains to the N-terminus of a nuclease-dead Cpfl enzyme from Acidaminococcus sp. BV3E6 containing a D908A mutation under control of the chicken beta-actin promoter and CMV enhancer sequence. The Fokl domains are separated from each other with a polypeptide linker and from the dCpfl with an XTEN linker.

FIG. 7 shows a schematic of an exemplary plasmid vector encoding a fusion polypeptide comprising a Cpfl domain lacking nuclease activity (dCpfl) and two Fokl nuclease domains (Fokl domain 1 and Fokl domain), wherein the Fokl domain 1 contains a D450A mutation abrogating its nuclease activity. The vector encodes a gRNA scaffold under the control of the U6 promoter. The fusion polypeptide is a fusion of the two Fokl nuclease domains to the C-terminus a nuclease-dead Cpfl enzyme from Acidaminococcus sp. BV3L6 containing a D908A mutation under control of the chicken beta-actin promoter and CMV enhancer sequence. The Fokl domains are separated from each other with a polypeptide linker and from the dCpf 1 with an XTEN linker.

FIG. 8 shows a schematic of the exemplary plasmid vector encoding a fusion polypeptide comprising a Cpfl domain lacking nuclease activity (dCpfl) and two Fokl nuclease domains (Fokl domain I and Fokl domain II), wherein the Fokl domain II contains a D450A mutation abrogating its nuclease activity. Fokl domain I contains the wildtype D450 residue and has functional nuclease activity. The vector encodes a gRNA scaffold under the control of the U6 promoter. The fusion polypeptide is a fusion of the two Fokl nuclease domains to the C-terminus of a nuclease-dead Cpfl enzyme from Acidaminococcus sp. BV3L6 containing a D908A mutation under control of the chicken beta-actin promoter and CMV enhancer sequence. The Fokl domains are separated from each other with a polypeptide linker and from the dCpfl with an XTEN linker.

FIG. 9 shows a schematic of an exemplary plasmid vector encoding a fusion polypeptide comprising a Cpfl domain lacking nuclease activity (dCpfl) and two Fokl nuclease domains (Fokl domain I and Fokl domain II), wherein Fokl domain I contains a D450A mutation abrogating its nuclease activity. Fokl domain II contains the wildtype D450 residue and has functional nuclease activity. The vector encodes a gRNA scaffold under the control of the U6 promoter. The fusion polypeptide is a fusion of the two Fokl nuclease domains to the N-terminus of a nuclease dead dCpfl enzyme from Acidaminococcus sp. BV3L6 (AsCpfl) containing a D908A mutation under control of the chicken beta-actin promoter and a CMV enhancer sequence. The Fokl domains are separated from each other with a polypeptide linker and from the dCpfl with an XTEN linker.

FIG. 10 shows a schematic of an exemplary plasmid vector encoding a fusion polypeptide comprising Cpfl domain lacking nuclease activity (dCpfl) and two Fokl nuclease domains (Fokl domain I and Fokl domain II), wherein the Fokl domain II has a D450A mutation abrogating its nuclease activity. The Fokl domain I contains the wildtype D450 residue and has functional nuclease activity. The vector encodes a gRNA scaffold under the control of the U6 promoter. The fusion polypeptide is a fusion of the two Fokl nuclease domains to the N-terminus of a nuclease dead (dCpfl) enzyme from Acidaminococcus sp. BV3L6 (AsCpfl) containing a D908A mutation under control of the chicken beta-actin promoter and a CMV enhancer sequence. The Fokl domains are separated from each other with a polypeptide linker and from the dCpfl with an XTEN linker.

FIG. 11 shows a schematic of an exemplary plasmid vector encoding a fusion polypeptide comprising base editing domain, a Cpfl domain lacking nuclease activity (dCpfl) and two FokI nuclease domains (FokI domain I and Fokl domain II), wherein FokI domain I has a D450A mutation abrogating its nuclease activity. The FokI domain II contains the wildtype D450 residue and has functional nuclease activity. The vector encodes a gRNA scaffold under the control of the U6 promoter. The fusion polypeptide is a fusion of the exemplary base editor APOBEC-1 to the N-terminus of the nuclease dead (dCpfl) enzyme from Acidaminococcus sp. BV3L6 (AsCpfl) containing a D908A mutation with the C-terminus of the dCpfl fused to the two FokI nuclease domains under the control of the chicken beta-actin promoter and a CMV enhancer sequence. The Fokl domains are separated from each other with a polypeptide linker and from the dCpfl with an XTEN linker.

FIG. 12 shows a schematic of the exemplary plasmid vector encoding a base editing domain, an enhanced nuclease based on MAD7™ nuclease, and two Fokl nuclease domains (Fokl domain I and Fokl domain II), wherein the Fokl domain I has a D450A mutation abrogating its nuclease activity. The Fokl domain II contains the wildtype D450 residue and has functional nuclease activity. The vector encodes a gRNA scaffold under the control of the U6 promoter. The fusion polypeptide is a fusion of the exemplary base editor APOBEC- 1 to the N-terminus of an enhanced catalytically dead nuclease based on MAD7™ containing mutations K169R, D529F, K535V, N538R, and D877A fused to the two Fokl nuclease domains under the control of the chicken beta-actin promoter and a CMV enhancer sequence. The Fokl domains are separated from each other with a polypeptide linker and from the dCpfl with an XTEN linker.

FIG. 13 shows a schematic of an exemplary fusion polypeptide described herein, wherein from N-terminus to C-terminus, the polypeptide comprises a base editor domain (e.g., TadA2.1), an endonuclease domain (e.g., Fokl nuclease domain), and a Cas domain lacking nuclease activity (dCas, e.g., Cas(|)/ Casl2j).

DETAILED DESCRIPTION

Aspects of the present disclosure provide fusion polypeptides comprising a Cpfl domain that is catalytically inactive (lacks nuclease activity) and an endonuclease domain (e.g., from a restriction endonuclease, such as FokI) that function in directing single stranded DNA cleavage (i.e., nickase activity) to a target site in the genome of a cell. In some embodiments, the fusion polypeptides further comprise a genomic modification domain, such as a base editor domain (e.g., a deaminase activity) that targets and deaminates a nucleobase, e.g., a cytosine or adenosine nucleobase of a C or A nucleotide, at the target site, which via cellular mismatch repair mechanisms, results in a modification, such as a change in the nucleobase from a C to a T nucleotide, or a change from an A to a G nucleotide.

Targeting of endonucleases to desired genomic target sites using transcription activator- like effector nucleases (TALENs) or zinc finger domains has been performed, and in the case of zinc finger nucleases (ZFNs), has been utilized to carry out genetic mutations (Ramirez, et al, Nucleic Acids Research (2012) 40 (12): 5560-68; Sun et al., Mol. BioSyst. (2014) 10: 446). However, generation of such constructs is laborious, may be cumbersome due to their large size (in the case of TALENS) and less efficient than genetic editing using CRISPR/Cas systems.

Precise genetic editing has been achieved, for example using base editors based primarily on a catalytically impaired Cas9 nuclease in which one of the nuclease domains of Cas9 is mutated such that the nuclease generates a single-strand DNA break. However, use of non-Cas9 nucleases, such as Casl2a/Cpfl nucleases for such genomic targeting has been much more limited. Without wishing to be bound by any particular theory, it is thought that in contrast to the two separate nuclease domains of Cas9, Casl2a/Cpfl does not have separate active sites for cleaving each DNA strand, making nickase variants of Casl2a/Cpfl more challenging. See, e.g., Richter et al. Nat. Biotechnol. (2020) 38(7): 883-891.

Fusion Polypeptides

Aspects of the present invention provide fusion polypeptides comprising a Cpfl/Casl2a domain without nuclease activity and an endonuclease domain, including systems and methods for using such fusion polypeptides for introducing targeted mutations into the genome of a target cell. The term “mutation,” as used herein, refers to a change (e.g., an insertion, deletion, inversion, or substitution) in a nucleic acid sequence as compared to a reference sequence, e.g., the corresponding sequence of a cell not having such a mutation, or the corresponding wild-type nucleic acid sequence.

In some embodiments, the cells produced using the fusion polypeptides described herein comprise more than one mutation (e.g., 2, 3, 4, 5, or more) mutations compared to a reference sequence, e.g., the corresponding sequence of a cell not having such a mutation, or the corresponding wild-type nucleic acid sequence. In some embodiments, a mutation to a gene (e.g., a target gene) results in a loss of expression of a protein encoded by the target gene in a cell harboring the mutation. In some embodiments, a mutation in a gene (e.g., a target gene) results in the expression of a variant form of a protein that is encoded by the target gene.

In some embodiments provided herein, the fusion polypeptides effect a mutation in a gene (e.g., a target gene) that results in a loss of expression of a protein encoded by the target gene in a cell harboring the mutation. In some embodiments, the fusion polypeptides effect a mutation in a gene (e.g., a target gene) results in the expression of a variant form of a protein that is encoded by the target gene. In some embodiments, a genetically engineered cell described herein is generated by using any of the fusion polypeptides described herein, for example under conditions suitable for the fusion polypeptide to be directed to target site in the genome of a cell (e.g., by a guide RNA (gRNA) described elsewhere herein) and for the endonuclease domain to cleave a phosphodiester bond in the DNA of the cell.

In some embodiments, the fusion polypeptides described herein generate genetically engineered cells via genome editing technology capable of introducing targeted changes, also referred to as “edits,” into the genome of a cell. In some embodiments, the genetically engineered cells comprise a plurality of edits in the genome of the cells.

The fusion polypeptides described herein comprise a Cpfl/Casl2a domain without nuclease activity and an endonuclease domain, and in some embodiments, may further comprises a genomic modification domain. In some embodiments, the fusion polypeptides comprise one or more linker domains, for example to join any of the domains of the polypeptide.

Cpfl domain

In some aspects, the present disclosure provides a CRISPR-Cas-based system for targeting a fusion polypeptide comprising a Cpfl domain lacking nuclease activity and an endonuclease domain to a genomic locus in a cell. As used herein, a “Cpfl domain” refers to Cpfl nuclease (also referred to as a Casl2 nuclease or Casl2a nuclease) or portion thereof or variant thereof. Cpfl is considered to belong to the class 2 type V-A Cas nucleases. See, e.g., Strohkendl et al. Mol. Cell (2018) 71: 1-9. The Casl2/Cpfl nucleases for use in the fusion polypeptides described herein refer to a polypeptide i) derived from a type II class 2 CRISPR/Cas nuclease that cleaves distal to a PAM site, and ii) capable of, in combination with a suitable gRNA, binding a target nucleic acid sequence (a target sequence). In contrast to Cas9 nucleases, Cpfl nucleases are directed to a target site requiring one gRNA molecule, a the CRISPR RNA (crRNA), rather than both a crRNA and tracrRNA sequence, and functions using a dual RuvC-Nuc domain (RuvC endonuclease and Nuc nuclease domain), whereas Cas9 has two nuclease domains (RuvC-Nuc and HNH). See, e.g., Gao et al. Cell Res. (2016) 26(8): 901-913.

In some embodiments, the Cpfl domain is a portion of a Cpfl enzyme comprising at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the Cpfl enzyme. In some embodiments, the Cpfl domain is one or more domains of a Cpfl enzyme.

Exemplary suitable Cpfl nucleases include, without limitation, AsCasl2a, FnCasl2a, LbCasl2a, PaCas 12a, other Cpfl orthologs, and Casl2a derivatives, such as the MAD7 system (MAD7™, Inscripta, Inc.), or the Alt-R Casl2a (Cpfl) Ultra nuclease (Alt-R® Casl2a Ultra; Integrated DNA Technologies, Inc.). See, e.g., Gill et al. LIPSCOMB 2017. In United States: Inscripta Inc.; Price et al. Biotechnol. Bioeng. (2020) 117(60): 1805-1816; PCT Publication Nos. WO 2016/166340; WO 2017/155407; WO 2018/083128; WO 2016/205711; WO 2017/035388; WO 2017/184768; WO2019/118516; WO2017/184768; WO 2018/098383; WO 2020/146297; and WO 2020/172502. In some embodiments, the Cpfl domain is from Casl2a/Cpfl obtained from Acidaminococcus sp. (referred to as “AsCasl2a” or “AsCpfl”), such as Acidaminococcus sp. strain BV3L6.

Additional examples of Casl2 nucleases for use in the fusion polypeptides described herein include, without limitation, Casl2g, Casl2c, Casl2d, Casl2e, Casl2i, Casl2h, Cas(|)/Casl2j and Casl2b.

Various Casl2/Cpfl nucleases are known in the art and may be obtained from various sources and/or engineered/modified to modulate one or more activities or specificities of the enzymes. For example, the PAM sequence preferences and specificities of a Casl2/Cpfl nucleases may be modified. In some embodiments, the Casl2/Cpfl nuclease has been engineered/modified to recognize one or more PAM sequence. In some embodiments, the Casl2/Cpfl nuclease has been engineered/modified to recognize one or more PAM sequence that is different than the PAM sequence the Casl2/Cpfl nuclease recognizes without engineering/modification. In some embodiments, the Casl2/Cpfl nuclease has been engineered/modified to reduce off-target activity of the enzyme.

In some embodiments, the Cpfl domain comprises an amino acid sequence of, or is derived from, a Cpfl protein from Prevotella spp., Francisella spp., Acidaminococcus sp. (AsCpfl), Lachnospiraceae bacterium (LpCpfl), Eubacterium rectale, or an engineered Cpfl. In some embodiments, the engineered Cpfl is the MAD7 system (MAD7™, Inscripta, Inc.). Amino acid sequences of exemplary Casl2/Cpfl nucleases are provided below.

Amino acid sequence of MAD7™ (Inscripta) (SEQ ID NO: 1)

MNNGTNNFQNFIGI SSLQKTLRNALIPTETTQQFIVKNGI IKEDELRGENRQILKDIMDDYYRGFI SETLSS IDD IDWTSLFEKMEIQLKNGDNKDTLIKEQTEYRKAIHKKFANDDRFKNMFSAKLISDILPEF VIHNNNYSASEKEEK TQVIKLFSRFATSFKDYFKNRANCFSADDISSSSCHRIVNDNAEIFFSNALVYRRIVKSL SNDDINKISGDMKDS LKEMSLEEIYSYEKYGEFITQEGISFYNDICGKVNSFMNLYCQKNKENKNLYKLQKLHKQ ILCIADTSYEVPYKF ESDEEVYQSVNGFLDNISSKHIVERLRKIGDNYNGYNLDKIYIVSKFYESVSQKTYRDWE TINTALEIHYNNILP GNGKSKADKVKKAVKNDLQKSITEINELVSNYKLCSDDNIKAETYIHEISHILNNFEAQE LKYNPEIHLVESELK ASELKNVLDVIMNAFHWCSVFMTEELVDKDNNFYAELEEIYDEIYPVISLYNLVRNYVTQ KPYSTKKIKLNFGIP TLADGWSKSKEYSNNAI ILMRDNLYYLGIFNAKNKPDKKI IEGNTSENKGDYKKMIYNLLPGPNKMIPKVFLSSK TGVETYKPSAYILEGYKQNKHIKSSKDFDITFCHDLIDYFKNCIAIHPEWKNFGFDFSDT STYEDISGFYREVEL QGYKIDWTYISEKDIDLLQEKGQLYLFQIYNKDFSKKSTGNDNLHTMYLKNLFSEENLKD IVLKLNGEAEIFFRK SS IKNP I IHKKGS ILVNRTYEAEEKDQFGNIQIVRKNIPENI YQELYKYFNDKSDKELSDEAAKLKNWGHHEAA TNIVKDYRYTYDKYFLHMPITINFKANKTGFINDRILQYIAKEKDLHVIGIDRGERNLIY VSVIDTCGNIVEQKS FNIVNGYDYQIKLKQQEGARQIARKEWKEIGKIKEIKEGYLSLVIHEI SKMVIKYNAI IAMEDLSYGFKKGRFKV ERQVYQKFETMLINKLNYLVFKDISITENGGLLKGYQLTYIPDKLKNVGHQCGCIFYVPA AYTSKIDPTTGFVNI FKFKDLTVDAKREFIKKFDSIRYDSEKNLFCFTFDYNNFITQNTVMSKSSWSVYTYGVRI KRRFVNGRFSNESDT IDITKDMEKTLEMTDINWRDGHDLRQDI IDYEIVQHIFEIFRLTVQMRNSLSELEDRDYDRLI SPVLNENNIFYD S AKAGDALPKDADANGAYC I ALKGLYE I KQ I TENWKEDGKF SRDKLKI SNKDWFDF I QNKRYL

Residues K169, D529, K535, N538, and D877 are indicated in boldface and underlined.

Variants of the MAD7™ sequence as provided above, or any suitable sequence of MAD™ known in the art (e.g., the sequence above without the N-terminal methionine, e.g., in the context of a fusion protein), are also embraced by the present disclosure. Such sequences include, for example, an MAD7™ sequence comprising an amino acid substitution at residue K169, D529, K535, N538, or D877, or two or more substitutions at any combination of these residues. In some embodiments, the MAD7™ sequence comprises an amino substitution at residue K169. In some embodiments, the amino acid substitution at residue K169 is a K169R substitution. In some embodiments, the MAD7™ sequence comprises an amino substitution at residue D529. In some embodiments, the amino acid substitution at residue D529 is a D529R substitution. In some embodiments, the MAD7™ sequence comprises an amino substitution at residue K535. In some embodiments, the amino acid substitution at residue K535 is a K535V substitution. In some embodiments, the MAD7™ sequence comprises an amino substitution at residue N538. In some embodiments, the amino acid substitution at residue N538 is a N538R substitution. In some embodiments, the MAD7™ sequence comprises an amino substitution at residue D877. In some embodiments, the amino acid substitution at residue D877 is a D877A substitution.

In some embodiments, the Cpfl domain comprises an amino acid sequence of SEQ ID NO: 1 that is lacking the N-terminal methionine, e.g., in the context of a fusion protein), are also embraced by the present disclosure. In some embodiments, the Cpfl domain comprises an amino acid sequence of SEQ ID NO: 1 comprising an amino acid substitution at residue K169, D529, K535, N538, or D877, or two or more substitutions at any combination of these residues. In some embodiments, the Cpfl domain comprises an amino acid sequence of SEQ ID NO: 1 and comprises an amino substitution at residue K169. In some embodiments, the amino acid substitution at residue K169 is a K169R substitution. In some embodiments, the Cpfl domain comprises an amino acid sequence of SEQ ID NO: 1 and comprises an amino substitution at residue D529. In some embodiments, the amino acid substitution at residue D529 is a D529R substitution. In some embodiments, the Cpfl domain comprises an amino acid sequence of SEQ ID NO: 1 and comprises an amino substitution at residue K535. In some embodiments, the amino acid substitution at residue

K535 is a K535V substitution, the Cpfl domain comprises an amino acid sequence of SEQ ID NO: 1 and comprises an amino substitution at residue N538. In some embodiments, the amino acid substitution at residue N538 is a N538R substitution. In some embodiments, the

Cpfl domain comprises an amino acid sequence of SEQ ID NO: 1 and comprises an amino substitution at residue D877. In some embodiments, the amino acid substitution at residue

D877 is a D877A substitution.

Amino acid sequence of MAD7™ (Inscripta) containing K169R, D529R, K535V, and

N538R mutations (SEQ ID NO: 2)

MNNGTNNFQNFIGI SSLQKTLRNALIPTETTQQFIVKNGI IKEDELRGENRQILKDIMDDYYRGFI SETLSS IDD IDWTSLFEKMEIQLKNGDNKDTLIKEQTEYRKAIHKKFANDDRFKNMFSAKLISDILPEF VIHNNNYSASEKEEK TQVIKLFSRFATSFKDYFRNRANCFSADDISSSSCHRIVNDNAEIFFSNALVYRRIVKSL SNDDINKISGDMKDS LKEMSLEEIYSYEKYGEFITQEGISFYNDICGKVNSFMNLYCQKNKENKNLYKLQKLHKQ ILCIADTSYEVPYKF ESDEEVYQSVNGFLDNISSKHIVERLRKIGDNYNGYNLDKIYIVSKFYESVSQKTYRDWE TINTALEIHYNNILP GNGKSKADKVKKAVKNDLQKSITEINELVSNYKLCSDDNIKAETYIHEISHILNNFEAQE LKYNPEIHLVESELK ASELKNVLDVIMNAFHWCSVFMTEELVDKDNNFYAELEEIYDEIYPVISLYNLVRNYVTQ KPYSTKKIKLNFGIP TLARGWSKSVEYSRNAI ILMRDNLYYLGIFNAKNKPDKKI IEGNTSENKGDYKKMIYNLLPGPNKMIPKVFLSSK TGVETYKPSAYILEGYKQNKHIKSSKDFDITFCHDLIDYFKNCIAIHPEWKNFGFDFSDT STYEDISGFYREVEL QGYKIDWTYISEKDIDLLQEKGQLYLFQIYNKDFSKKSTGNDNLHTMYLKNLFSEENLKD IVLKLNGEAEIFFRK SS IKNP I IHKKGS ILVNRTYEAEEKDQFGNIQIVRKNIPENI YQELYKYFNDKSDKELSDEAAKLKNWGHHEAA TNIVKDYRYTYDKYFLHMPITINFKANKTGFINDRILQYIAKEKDLHVIGIDRGERNLIY VSVIDTCGNIVEQKS FNIVNGYDYQIKLKQQEGARQIARKEWKEIGKIKEIKEGYLSLVIHEI SKMVIKYNAI IAMEDLSYGFKKGRFKV ERQVYQKFETMLINKLNYLVFKDISITENGGLLKGYQLTYIPDKLKNVGHQCGCIFYVPA AYTSKIDPTTGFVNI FKFKDLTVDAKREFIKKFDSIRYDSEKNLFCFTFDYNNFITQNTVMSKSSWSVYTYGVRI KRRFVNGRFSNESDT IDITKDMEKTLEMTDINWRDGHDLRQDI IDYEIVQHIFEIFRLTVQMRNSLSELEDRDYDRLI SPVLNENNIFYD S AKAGDALPKDADANGAYC I ALKGLYE I KQ I TENWKEDGKF SRDKLKI SNKDWFDF I QNKRYL

Amino acid sequence of MAD7™ (Inscripta) containing K169R, D529R, K535V, N538R, and D877A mutations (SEQ ID NO: 3)

MNNGTNNFQNFIGI SSLQKTLRNALIPTETTQQFIVKNGI IKEDELRGENRQILKDIMDDYYRGFI SETLSS IDD IDWTSLFEKMEIQLKNGDNKDTLIKEQTEYRKAIHKKFANDDRFKNMFSAKLISDILPEF VIHNNNYSASEKEEK TQVIKLFSRFATSFKDYFRNRANCFSADDISSSSCHRIVNDNAEIFFSNALVYRRIVKSL SNDDINKISGDMKDS LKEMSLEEIYSYEKYGEFITQEGISFYNDICGKVNSFMNLYCQKNKENKNLYKLQKLHKQ ILCIADTSYEVPYKF ESDEEVYQSVNGFLDNISSKHIVERLRKIGDNYNGYNLDKIYIVSKFYESVSQKTYRDWE TINTALEIHYNNILP GNGKSKADKVKKAVKNDLQKSITEINELVSNYKLCSDDNIKAETYIHEISHILNNFEAQE LKYNPEIHLVESELK ASELKNVLDVIMNAFHWCSVFMTEELVDKDNNFYAELEEIYDEIYPVISLYNLVRNYVTQ KPYSTKKIKLNFGIP TLARGWSKSVEYSRNAI ILMRDNLYYLGIFNAKNKPDKKI IEGNTSENKGDYKKMIYNLLPGPNKMIPKVFLSSK TGVETYKPSAYILEGYKQNKHIKSSKDFDITFCHDLIDYFKNCIAIHPEWKNFGFDFSDT STYEDISGFYREVEL QGYKIDWTYISEKDIDLLQEKGQLYLFQIYNKDFSKKSTGNDNLHTMYLKNLFSEENLKD IVLKLNGEAEIFFRK SS IKNP I IHKKGS ILVNRTYEAEEKDQFGNIQIVRKNIPENI YQELYKYFNDKSDKELSDEAAKLKNWGHHEAA TNIVKDYRYTYDKYFLHMPITINFKANKTGFINDRILQYIAKEKDLHVIGIARGERNLIY VSVIDTCGNIVEQKS FNIVNGYDYQIKLKQQEGARQIARKEWKEIGKIKEIKEGYLSLVIHEI SKMVIKYNAI IAMEDLSYGFKKGRFKV ERQVYQKFETMLINKLNYLVFKDISITENGGLLKGYQLTYIPDKLKNVGHQCGCIFYVPA AYTSKIDPTTGFVNI FKFKDLTVDAKREFIKKFDSIRYDSEKNLFCFTFDYNNFITQNTVMSKSSWSVYTYGVRI KRRFVNGRFSNESDT IDITKDMEKTLEMTDINWRDGHDLRQDI IDYEIVQHIFEIFRLTVQMRNSLSELEDRDYDRLI SPVLNENNIFYD S AKAGDALPKDADANGAYC I ALKGLYE I KQ I TENWKEDGKF SRDKLKI SNKDWFDF I QNKRYL

Exemplary amino acid sequence of Cpfl from Acidaminococcus sp. (SEQ ID NO: 4) corresponding to Uniprot Accession No. U2UMQ6.

MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKP I IDRI YKTYADQCLQLVQLDWEN LSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKA ELFNGKVLKQLGTVT TTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTR LITAVPSLREHFENV KKAIGIFVSTS IEEVFSFPFYNQLLTQTQIDLYNQLLGGI SREAGTEKIKGLNEVLNLAIQKNDETAHI IASLPH RFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSID LTHIFISHKKLETIS SALCDHWDTLRNALYERRI SELTGKITKSAKEKVQRSLKHEDINLQEI I SAAGKELSEAFKQKTSEILSHAHAAL DQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPS LSFYNKARNYATKKP YSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEK TSEGFDKMYYDYFPD AAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYA KKTGDQKGYREALCK WIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAV ETGKLYLFQIYNKDF AKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKK LKDQKTPIPDTLYQE LYDYVNHRLSHDLSDEARALLPNVITKEVSHEI IKDRRFTSDKFFFHVP ITLNYQAANSPSKFNQRVNAYLKEHP ETP I IGIDRGERNLI YITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSWGTIKDLKQGYLSQV IHEIVDLMIHYQAVWLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKV GGVLNPYQLTDQFT SFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKT GDFILHFKMNRNLSF QRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIAL LEEKGIVFRDGSNIL PKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPM DADANGAYHIALKGQ LLLNHLKESKDLKLQNGI SNQDWLAYIQELRN

Residues E174, S542, K548, N552, and D908 are indicated in boldface and underlined.

Variants of the Cpfl sequence as provided above, or any suitable sequence of Cpfl known in the art (e.g., the sequence above without the N-terminal methionine, e.g., in the context of a fusion protein), are also embraced by the present disclosure. Such sequences include, for example, a Cpfl sequence comprising an amino acid substitution at residue E174, S542, K548, N552, and D908, or two or more substitutions at any combination of these residues. In some embodiments, the Cpfl sequence comprises an amino substitution at residue E174. In some embodiments, the amino acid substitution at residue E174 is a E174R substitution. In some embodiments, the Cpfl sequence comprises an amino substitution at residue S542. In some embodiments, the amino acid substitution at residue S542 is a S542R substitution. In some embodiments, the Cpfl sequence comprises an amino substitution at residue K548. In some embodiments, the amino acid substitution at residue K548 is a K548V substitution. In some embodiments, the Cpfl sequence comprises an amino substitution at residue N552. In some embodiments, the amino acid substitution at residue N552 is a N552R substitution. In some embodiments, the Cpfl sequence comprises an amino substitution at residue D908. In some embodiments, the amino acid substitution at residue D908 is a D908A substitution.

In some embodiments, the Cpfl domain comprises an amino acid sequence of SEQ ID NO: 4 that is lacking the N-terminal methionine, e.g., in the context of a fusion protein), are also embraced by the present disclosure. In some embodiments, the Cpfl domain comprises an amino acid sequence of SEQ ID NO: 4 comprising an amino acid substitution at residue E174, S542, K548, N552, and D908, or two or more substitutions at any combination of these residues. In some embodiments, the Cpfl domain comprises an amino acid sequence of SEQ ID NO: 4 and comprises an amino substitution at residue E174. In some embodiments, the amino acid substitution at residue E174 is a E174R substitution. In some embodiments, the Cpfl domain comprises an amino acid sequence of SEQ ID NO: 4 and comprises an amino substitution at residue S542. In some embodiments, the amino acid substitution at residue S542 is a S542R substitution. In some embodiments, the Cpfl domain comprises an amino acid sequence of SEQ ID NO: 4 and comprises a substitution at residue K548. In some embodiments, the amino acid substitution at residue K548 is a K548V substitution. In some embodiments, the Cpfl domain comprises an amino acid sequence of SEQ ID NO: 4 and comprises an amino substitution at residue N552. In some embodiments, the amino acid substitution at residue N552 is a N552R substitution. In some embodiments, the Cpfl domain comprises an amino acid sequence of SEQ ID NO: 4 and comprises an amino substitution at residue D908. In some embodiments, the amino acid substitution at residue D908 is a D908A substitution.

Exemplary amino acid sequence of Cpfl from Acidaminococcus sp. containing E174R, S542R, K548V, N552R, and D908A mutations (SEQ ID NO: 5) TQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGF IEEDKARNDHYKELKP I IDRI YKTYADQCLQLVQLDWENL SAAIDSYRKEKTEETRNALIEEQATYRNAIHDYF IGRTDNLTDAINKRHAE IYKGLFKAELFNGKVLKQLGTVTT TEHENALLRSFDKFTTYFSGFYRNRKNVFSAED I STAIPHRIVQDNFPKFKENCHIFTRLI TAVP SLREHFENVK KAIGIFVSTS IEEVFSFPFYNQLLTQTQIDLYNQLLGGI SREAGTEKIKGLNEVLNLAIQKNDETAHI IASLPHR F IPLFKQILSDRNTLSF ILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNS IDLTHIF I SHKKLET I S S ALCDHWDTLRNALYERRI SELTGKI TKSAKEKVQRSLKHED INLQE I I SAAGKELSEAFKQKTSE ILSHAHAALD QPLPTTLKKQEEKE ILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEP SLSFYNKARNYATKKPY SVEKFKLNFQMPTLARGWDVNVEKNRGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKT SEGFDKMYYDYFPDA AKMIPKCSTQLKAVTAHFQTHTTP ILLSNNF IEPLE I TKE IYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKW IDFTRDFLSKYTKTTS IDLS SLRP S SQYKDLGEYYAELNPLLYHI SFQRIAEKE IMDAVETGKLYLFQIYNKDFA KGHHGKPNLHTLYWTGLFSPENLAKTS IKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTP IPDTLYQEL YDYVNHRLSHDLSDEARALLPNVI TKEVSHE I IKDRRFTSDKFFFHVP I TLNYQAANSP SKFNQRVNAYLKEHPE TP I IGIARGERNLI YI TVIDSTGKILEQRSLNT IQQFDYQKKLDNREKERVAARQAWSWGT IKDLKQGYLSQVI HE IVDLMIHYQAVWLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGV LNPYQLTDQFTS FAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKT IKNHESRKHFLEGFDFLHYDVKTGDF ILHFKMNRNLSFQ RGLPGFMPAWD IVFEKNETQFDAKGTPF IAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILP KLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMD ADANGAYHIALKGQL LLNHLKESKDLKLQNGI SNQDWLAYIQELRN Exemplary amino acid sequence of Cpfl from Prevotella spp. (SEQ ID NO: 6) corresponding to Uniprot Accession No. A0A350PSL0.

MAKNFEDFKRLYPLSKTLRFEAKPIGATLDNIVKSGLLEEDEHRAASYVKVKKLIDE YHKVFIDRVLDNGCLPLD DKGDNNSLAEYYESYVSKAQDEDAIKKFKEIQQNLLS I IAKKLTDDKAYANLFGNKLIESYKDKADKTKLIDSDL IQFINTAESTQLVSMSQDEAKELVKEFWGFTTYFEGFFKNRKNMYTPEEKSTGIAYRLIN ENLPKFIDNMEAFKK AIARPEIQANMEELYSNFSEYLNVES IQEMFLLDYYNMLLTQKQIDVYNAI IGGKTDDEHDVKIKGINEYINLYN QQHKDDKLPKLKALFKQILSDRNAISWLPEEFNSDQEVLNAIKDCYERLAENVLGDKVLK SLLGSLADYSLDGIF IRNDLQLTDISQKMFGNWGVIQNAIMQNIKHVAPARKHKESEEDYEKRIAGIFKKADSFS ISYINDCLNEADPNN AYFVENYFATFGAVNTPTMQRENLFALVQNAYTEVAALLHSDYPTVKHLAQDKANVSKIK ALLDAIKSLQHFVKP LLGKGDESDKDERFYGELASLWAELDTVTPLYNMIRNYMTRKPYSQKKIKLNFENPQLLG GWDANKEKDYATI IL RRNGLYYLAIMDKDSRKLLGKAMPSDGECYEKMVYKFFKDVTTMIPKCSTQLKDVQAYFK VNTDDYVLNSKAFNR PLTITKEVFDLNNVLYGKYKKFQKGYLTATGDNVGYTHAVNVWIKFCMDFLDSYDSTCIY DFSSLKPESYLSLDS FYQDVNLLLYKLSFTDVSASFIDQLVEEGKMYLFQIYNKDFSEYSKGTPNMHTLYWKALF DERNLADWYKLNGQ AEMFYRKKSIENTHPTHPANHPILNKNKDNKKKESLFEYDLIKDRRYTVDKFMFHVPITM NFKSSGSENINQDVK AYLRHADDMHI IGIDRGERHLLYLWIDLQGNIKEQFSLNEIVNDYNGNTYHTNYHDLLDVREDERLKARQS WQT IENIKELKEGYLSQVIHKITQLMVRYHAIWLEDLSKGFMRSRQKVEKQVYQKFEKMLIDK LNYLVDKKTDVSTP GGLLNAYQLTCKSDSSQKLGKQSGFLFYIPAWNTSKIDPVTGFVNLLDTHSLNSKEKIKA FFSKFDAIRYNKDKK WFEFNLDYDKFGKKAEDTRTKWTLCTRGMRIDTFRNKEKNSQWDNQEVDLTTEMKSLLEH YYIDIHGNLKDAIST QTDKAFFTGLLHILKLTLQMRNSITGTETDYLVSPVADENGIFYDSRSCGDQLPENADAN GAYNIARKGLMLVEQ IKDAEDLDNVKFDISNKAWLNFAQQKPYKNG

Exemplary amino acid sequence of Cpfl from Francisella spp. (SEQ ID NO: 7) corresponding to Uniprot Accession No. A0Q7Q2.

MS I YQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQI IDKYHQFFIEEILSSVCI SEDL LQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQE SDLILWLKQSKDNGI ELFKANSD I TD IDEALEI IKSFKGWTTYFKGFHENRKNVYSSNDIPTS I I YRIVDDNLPKFLENKAKYESLKDKA PEAINYEQIKKDLAEELTFDIDYKTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTI IGGKFVNGENTKRKGI NEYINLYSQQINDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDWTTMQSFYEQIAA FKTVEEKSIKETLS LLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSK KEQELIAKKTEKAKY LSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQG KKDLLQASAEDDVKA IKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNY ITQKPYSDEKFKLNF ENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYK LLPGANKMLPKVFFS AKSIKFYNPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWK DFGFRFSDTQRYNSI DEFYREVENQGYKLTFENISESYIDSWNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKAL FDERNLQDWYKLN GEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPIT INFKSSGANKFNDEI NLLLKEKANDVHILS IDRGERHLAYYTLVDGKGNI IKQDTFNI IGNDRMKTNYHDKLAAIEKDRDSARKD WKKIN NIKEMKEGYLSQWHEIAKLVIEYNAIWFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLNY LVFKDNEFDKTGG VLRAYQLTAPFETFKKMGKQTGI I YYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFE FSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEY GHGECIKAAICGESD KKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDADANGAY HIGLKGLMLLGRIKN NQEGKKLNLVIKNEEYFEFVQNRNN

Exemplary amino acid sequence of Cpfl from Lachnospiraceae spp. (SEQ ID NO: 8) corresponding to Uniprot Accession No. A0A7C9H0Z9.

MNGNRI IVYREFVGVTPVAKTLRNELRP IGHTQEHI IHNGLIQEDELRQEKSTELKNIMDDYYREYIDKSLSGVT DLDFTLLFELMNLVQSSPSKDNKKALEKEQSKMREQICTHMQSDSNYKNIFNAKFLKEIL PDFIKNYNQYDAKDK AGKLETLALFNGFSTYFTDFFEKRKNVFTKEAVSTSIAYRIVHENSLTFLANMTSYKKIS EKALDEIEVIEKNNQ DKMGDWELNQIFNPDFYNMVLIQSGIDFYNEICGWNAHMNLYCQQTKNNYNLFKMRKLHK QILAYTSTSFEVPK MFEDDMSVYNAVNAFIDETEKGNI IGKLKDIVNKYDELDEKRI YI SKDFYETLSCFMSGNWNLITGCVENFYDEN IHAKGKSKEEKVKKAVKEDKYKS INDVNDLVEKYIDEKERNEFKNSNAKQYIREI SNI ITDTETAHLEYDEHI SL IESEEKADEMKKRLDMYMNMYHWAKAFIVDEVLDRDEMFYSDIDDIYNILENIVPLYNRV RNYVTQKPYNSKKIK LNFQSPTLANGWSQSKEFDNNAI ILIRDNKYYLAIFNAKNKPDKKI IQGNSDKKNDNDYKKMVYNLLPGANKMLP KVFLSKKGIETFKPSDYI I SGYNAHKHIKTSENFDI SFCRDLIDYFKNS IEKHAEWRKYEFKFSATDSYNDI SEE YREVEMQGYRIDWTYI SEADINKLDEEGKI YLFQI YNKDFAENSTGKENLHTMYFKNIFSEENLKDI I IKLNGQA ELF YRRAS VKNP VKHKKD S VLVNKT YKNQLDNGD WRI P I PDD I YNE I YKMYNGY I KE SDLSGAAKE YLDKVEVR TAQKEIVKDYRYTVDKYFIHTPITINYKVAARNNVNDMAVKYIAQNDDIHVIGIDRGERN LIYISVIDSHGNIVK QKSYNILNNYDYKKKLVEKEKTREYARKNWKS IGNIKELKEGYI SGWHEIAMLMVEYNAI IAMEDLNYGFKRGR FKVERQVYQKFESMLINKLNYFASKGKSVDEPGGLLKGYQLTYVPDNIKNLGKQCGVIFY VPAAFTSKIDPSTGF ISAFNFKSISTNDSRKQFFMQFDEIRYCAEKDMFSFGFDYNNFDTYNITMGKTQWTVYTN GERLQSEFNNARRTG KTKSINLTETIKLLLEDNEINYADGHDVRIDMEKMDEDKNSEFFAQLLSLYKLTVQMRNS YTEAEEQEKGISYDK I I SPVINDEGEFFDSDNYKESDDKECKMPKDADANGAYCIALKGLYEVLKIKSEWTEDGFDR NCLKLPHAEWLDF IQNKRYE

Exemplary amino acid sequence of Cpfl from Eubacterium rectale (SEQ ID NO: 9) corresponding to Uniprot Accession No. A0A6L5T656.

MNNGTNNFQNFIGI SSLQKTLRNALIPTETTQQFIVKNGI IKEDELRGENRQILKDIMDDYYRGFI SELLS S IDD IDWTSLFEKMEIQLKNGDNKDTLIKEQAEKRKAIYKKFADDDRFKNMFSAKLISDILEFV IHNNNYSASEKEEKT QVIKLFSRFATSFKDYFKNRANCFSADDISSSSCHRIVNDNAEIFFSNALVYRRIVKNLS NDDINKISGDMKDSL KEMSLDEIYSYEKYGEFITQEGISFYNDICGKVNSFMNLYCQKNKENKNLYKLRKLHKQI LCIADTSYEVPYKFE SDEEVYQSVNGFLDNI S SKHI VERLRKIGDNYNGYNLDKI YI VSRFYESVSQKTYRD WET INTALE IHYNNILPG NGKSKADKVKKAVKNDLQKSITEINELVSNYKLCPDDNIKAETYIHEISHILNNFEAQEL KYNPEIHLVESELKA SELKNVLDVIMNAFHWCSVFMTEELVDKDNNFYAELEEIYDEIYPVISLYNLVRNYVTQK PYSTKKIKLNFGIPT LADGWSKSKEYSNNAI ILMRDNLYYLGIFNAKNKPDKKI IEGNTSENKGDYKKMIYNLLPGPNKMIPKVFLSSKT GVETYKPSAYILEGYKQNKHLKSSKDFDITFCRDLIDYFKNCIAIHPEWKNFGFDFSDTS TYEDISGFYREVELQ GYKIDWTYISEKDIDLLQEKGQLYLFQIYNKDFSKKSTGNDNLHTMYLKNLFSEENLKDI VLKLNGEAEIFFRKS S IKNP I IHKKGS ILVNRTYEAEEKDQFGNIQIVRKTIPENI YQELYKYFNDKSDKELSDEAAKLKNWGHHEAAT NIVKDYRYTYDKYFLHMPITINFKANKTSFINDRILQYIAKEKDLHVIGIDRGERNLIYV SVIDICGNIVEQKSF NIVNGYDYQIKLKQQEGARQIARKEWKEIGKIKEIKEGYLSLVIHEI SKMVIKYNAI IAMEDLSYGFKKGRFKVE RQVYQKFETMLINKLNYLVFKDISITENGGLLKGYQLTYIPDKLKNVGHQCGCIFYVPAA YTSKIDPTTGFVNIF KFKDLTVDAKREFIKKFDSIRYDSDKNLFCFTFDYNNFITQNTVMSKSSWSVYTYGVRIK RRFVNGRFSNESDTI DITKDMEKTLEMTDINWRDGHDLRQDI IDYEIVQHIFEIFKLTVQMRNSLSELEDRDYDRLI SPVLNENNIFYDS AKAGDALPKDADANGAYC I ALKGLYE I KQ I TENWKEDGKF SRDKLKI SNKDWFDF I QNKRYL

Both naturally occurring and modified variants of Cpfl enzymes are suitable for use according to aspects of this disclosure. For example, in some embodiments, a Cpfl domain is modified to reduce or eliminate nuclease activity of the domain. A catalytically inactive Cas nuclease may be referred to as “dead Cas 12” “dCasl2,” “dead Cpfl,” or “dCpfl.” In some embodiments, the inactive Cas nuclease is “dead Cas$” or “dCas$.” To generate a Cpfl domain lacking nuclease activity, any mutation (e.g., an insertion, deletion, inversion, or substitution) of one or more amino acids of the Cpfl may be made such that the nuclease activity is reduced as compared to a Cpfl domain that does contain the mutation (e.g., a wildtype Cpfl domain). In some embodiments, the Cpfl domain does not have detectable nuclease activity. Exemplary mutations that reduce or eliminate nuclease activity of the Cpfl enzyme are known in the art. See, e.g., Liu et al. Nature Communications (2017) 8: 2095. In some embodiments, the Cpfl domain comprises a mutation of an amino acid residue corresponding to the aspartic acid residue at position 908 (referred to as “D908”) of Cpfl from Acidaminococcus sp. (AsCpfl). In some embodiments, the Cpfl domain comprises a mutation of an amino acid residue corresponding to the aspartic acid residue at position 908 (referred to as “D908”) of Cpfl from Acidaminococcus sp. (AsCpfl) provided by SEQ ID NO: 4. In some embodiments, the Cpfl domain comprises a substitution of an amino acid residue corresponding to the aspartic acid residue at position 908 of Cpfl from Acidaminococcus sp. (AsCpfl) provided by SEQ ID NO: 4, to any other amino acid residue other than aspartic acid. In some embodiments, the Cpfl domain comprises a substitution of an amino acid residue corresponding to the aspartic acid residue at position 908 of Cpfl from Acidaminococcus sp. (AsCpfl) provided by SEQ ID NO: 4, to an alanine residue (referred to as “D908A”). In some embodiments, to generate a dead Case}) lacking nuclease activity, the Cas$ protein is engineered to comprise D371A and D394A in the RuvC domain (see, e.g., Pausch et al. Science. (2020) 369: 333-337, incorporated by reference in its entirety).

In some embodiments, the Cpfl domain is based on the MAD7™ enzyme (Inscripta). In such embodiments, an exemplary mutation that results in reduction or elimination of nuclease activity of the enzyme comprises a substitution of the aspartic acid residue at position 877 of MAD7™ provided by SEQ ID NO: 1, to any other amino acid residue other than aspartic acid. In some embodiments, the Cpfl domain is based on the MAD7™ enzyme (Inscripta) and comprises a substitution of the aspartic acid residue at position 877 of MAD7™ provided by SEQ ID NO: 1, to an alanine residue (referred to as “D877A”).

In some embodiments, the Cpfl domain comprises one or more mutations, for example to modulate genome editing activity, modulate editing efficiency, and/or reduce off target effects. See, e.g., Kleinstiver et al. Nature Biotech. (2019) 37: 276-282, incorporated by reference in its entirety. In some embodiments, the Cpfl domain comprises one or more mutations relative to a corresponding wildtype Cpfl nuclease. In some embodiments, the Cpfl domain comprises one or more substitutions in the Cpfl domain relative to a corresponding wildtype Cpfl domain.

In some embodiments, the Cpfl domain comprises a substitution of at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acids of the Cpfl domain relative to a corresponding wildtype Cpfl domain. In some embodiments, the Cpfl domain comprises a substitution of an amino acid at: one, two, three, or each of amino acids corresponding to positions 174, 542, 548, or 552 of the Acidaminococcus sp. Cpfl amino acid sequence (referred to as E174, S542, K548, and N552). In some embodiments, the Cpfl domain comprises a substitution of an amino acid residue corresponding to the glutamic acid at position 174 of Cpfl from Acidaminococcus sp, to any other amino acid residue other than glutamic acid. In some embodiments, the Cpfl domain comprises a substitution of an amino acid residue corresponding to the glutamic acid at position 174 of Cpfl from Acidaminococcus sp, to an arginine residue (E174R). In some embodiments, the Cpfl domain comprises a substitution of an amino acid residue corresponding to the serine at position 542 of Cpf 1 from Acidaminococcus sp, to any other amino acid residue other than serine. In some embodiments, the Cpfl domain comprises a substitution of an amino acid residue corresponding to the serine at position 542 of Cpfl from Acidaminococcus sp, to an arginine residue (S542R). In some embodiments, the Cpfl domain comprises a substitution of an amino acid residue corresponding to the lysine at position 548 of Cpfl from Acidaminococcus sp, to any other amino acid residue other than lysine. In some embodiments, the Cpfl domain comprises a substitution of an amino acid residue corresponding to the lysine at position 548 of Cpfl from Acidaminococcus sp, to a valine residue (K548V). In some embodiments, the Cpfl domain comprises a substitution of an amino acid residue corresponding to the asparagine at position 552 of Cpfl from Acidaminococcus sp, to any other amino acid residue other than asparagine. In some embodiments, the comprises a substitution of an amino acid residue corresponding to the asparagine at position 552 of Cpfl from Acidaminococcus sp, to a arginine residue (N552R).

In some embodiments, the Cpfl domain comprises a substitution of an amino acid residue corresponding to each of positions 174, 542, 548, and 552 of Cpfl from Acidaminococcus sp, to any other amino acid residue. In some embodiments, the Cpfl domain comprises a substitution mutation corresponding to each of E174R, S542R, K548V, and N552R.

In some embodiments, the Cpfl domain comprises a substitution of at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acids of the Cpfl domain relative to a corresponding wildtype MAD7™ Cpfl amino acid sequence. In some embodiments, the Cpfl domain comprises a substitution of an amino acid at: one, two, three, or each of amino acids corresponding to positions 169, 529, 535, or 538 of the MAD7™ Cpfl amino acid sequence (referred to as E169, D529, K535, and N538).

In some embodiments, the Cpfl domain comprises a substitution of an amino acid residue corresponding to the glutamic acid at position 169 of the MAD7™ Cpfl amino acid sequence, to any other amino acid residue other than glutamic acid. In some embodiments, the Cpfl domain comprises a substitution of an amino acid residue corresponding to the glutamic acid at position 169 of MAD7™ Cpfl amino acid sequence to an arginine residue (E169R). In some embodiments, the Cpfl domain comprises a substitution of an amino acid residue corresponding to the aspartic acid at position 529 of the MAD7™ Cpfl amino acid sequence, to any other amino acid residue other than aspartic acid. In some embodiments, the Cpfl domain comprises a substitution of an amino acid residue corresponding to the aspartic acid at position 529 of MAD7™ Cpfl amino acid sequence to an arginine residue (D529R). In some embodiments, the Cpfl domain comprises a substitution of an amino acid residue corresponding to the lysine at position 535 of the MAD7™ Cpfl amino acid sequence, to any other amino acid residue other than lysine. In some embodiments, the Cpfl domain comprises a substitution of an amino acid residue corresponding to the lysine at position 535 of MAD7™ Cpfl amino acid sequence to a valine residue (K535V). In some embodiments, the Cpfl domain comprises a substitution of an amino acid residue corresponding to the asparagine at position 538 of the MAD7™ Cpfl amino acid sequence, to any other amino acid residue other than asparagine. In some embodiments, the Cpfl domain comprises a substitution of an amino acid residue corresponding to the asparagine at position 538 of MAD7™ Cpfl amino acid sequence to an arginine residue (N538R).

In some embodiments, the Cpfl domain comprises a substitution of an amino acid residue corresponding to each of positions 169, 529, 535, or 538 of MAD7™ Cpfl amino acid sequence to any other amino acid residue. In some embodiments, the Cpfl domain comprises a substitution mutation corresponding to each of K169, D529R, K535V, and N538R.

In some embodiments, the amino acid sequence of the first Cpfl protein comprises any of SEQ ID NOs: 1-9 or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity or higher to any thereof. In some embodiments, the amino acid sequence of the second Cpfl protein comprises any of SEQ ID NOs: 1-9 or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity or higher to any thereof. In some embodiments, the chimeric polypeptide comprises an amino acid sequence of any of SEQ ID NOs: 1-9 or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity or higher to any thereof.

Endonuclease Domain

The fusion polypeptides described herein comprise a Cpfl domain that lacks nuclease activity and an endonuclease domain. In some embodiments, the fusion polypeptides comprise a Cpfl domain that lacks nuclease activity, an endonuclease domain, and a genomic modification domain. As used herein, an “endonuclease domain” refers to an enzyme, or portion thereof, that is capable of cleaving a phosphodiester bond between two nucleotides, resulting in a single or double stranded break in the polynucleotide. In general, endonucleases may cleave between two nucleotides in a sequence-specific or a sequenceindependent manner. In some embodiments, the endonuclease cleaves a phosphodiester bond between two nucleotides following recognition of a particular nucleotide sequence (/'.<?., a recognition site). In some embodiments, endonucleases that cleave between two nucleotides in a sequence-specific manner may be referred to as restriction enzymes or restriction endonucleases.

Endonucleases are typically categorized based on factors, such as the structure of the recognition site, position of cleavage relative to the recognition site, and whether endonuclease activity requires the presence of any enzyme cofactors. Examples of types of endonucleases include, Type 1 endonucleases, Type II endonucleases, Type III endonucleases, Type IV endonucleases, and Type V endonucleases.

In some embodiments, fusion polypeptides described herein comprise a Type II endonuclease or a domain thereof. Type II endonucleases form a homodimer and recognize and cleave nucleic acid at a position near (e.g., within 1, 2, 3, 4, or 5 nucleotides of the recognition site) or within the recognition site, resulting in a double stranded break of the polynucleotide. Subtypes of Type II endonucleases include, Type IIA, Type IIB, Type IIC, Type IIE, Type IIF, Type IIG, Type IIH, Type IIM, Type IIP, Type IIS, and Type IIT. See, e.g., Pingoud et al. Nucleic Acids Research (2014) 42(12): 7489-7527.

In some embodiments, the fusion polypeptides described herein comprise an endonuclease domain of a restriction endonuclease, such as a Type II endonuclease.

In some embodiments, fusion polypeptides described herein comprise a Type IIS endonuclease or a portion thereof. Type IIS restriction enzymes are characterized as being comprised of more than one subunit: a subunit comprising a DNA-binding domain and a subunit comprising a DNA-cleavage domain. Without wishing to be bound by any particular theory, it is generally thought that Type IIS endonucleases interact with a particular recognition site through the DNA-binding domain, form homodimers, and cleave the phosphodiester bond between two nucleotides near the recognition site. Non-limiting examples of Type IIS restriction enzymes include FokI, Acul, Alwl, Bael, BbsI, BbsI-HF, Bbvl, Bed, BceAI, Bcgl, BclVI, BcoDI, Bfil, BfuAI, BmrI, Bpml, BpuEI, BsaI-HFv2, BsaXI, BseRI, Bsgl, BsmAI, BsmBI-v2, BsmFI, BsmI, BspCNI, BspMI, BspQI, BsrDI, BsrI, BtgZI, BtsCI, Btsl-v2, BtsIMutl, CspCI, Earl, Ecil, Esp3I, Faul, Hgal, HphI, HpyAV, MboII, Mlyl, Mmel, Mnll, NmeAIII, PaqCI, Piel, SapI, and SfaNI. In some embodiments, the fusion polypeptides described herein comprise a Fokl endonuclease or a portion thereof.

In some embodiments, the endonuclease domain is a portion of a restriction endonuclease comprising at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the restriction endonuclease enzyme. In some embodiments, the endonuclease domain is one or more domains of a restriction endonuclease, such as a DNA-cleavage domain, a dimerization domain, and a catalytic site.

In some embodiments, the endonuclease domain comprises a first DNA-cleavage domain that is capable of forming a dimer with a second DNA-cleavage domain, which may have the same amino acid sequence as the first DNA-cleavage domain, or a different amino acid sequence as compared to the first DNA-cleavage domain. In some embodiments, the endonuclease domain of the fusion polypeptide comprises a first DNA-cleavage domain is capable of forming a dimer with a second DNA-cleavage domain. In some embodiments, the endonuclease domain of the fusion polypeptide comprises a first DNA-cleavage domain is capable of forming a dimer with a second DNA-cleavage domain that is present in a separate polypeptide. In some embodiments, the endonuclease domain of the fusion polypeptide comprises a first DNA-cleavage domain and a second DNA-cleavage domain, wherein the first DNA-cleavage domain and second DNA-cleavage domain are capable of forming a dimer with one another (e.g., within the same fusion polypeptide). In some embodiments, the endonuclease domain of the fusion polypeptide does not include a DNA-binding domain of a restriction endonuclease.

In some embodiments, a dimer of the first DNA-cleavage domain and second DNA- cleavage domain generates a double- stranded break in a targeted polynucleotide. In some embodiments, a dimer of the first DNA-cleavage domain and second DNA-cleavage domain generates a double- stranded break in a targeted polynucleotide. Such single- stranded break activity may be referred to as a “nickase.” In some embodiments, a dimer of the first DNA- cleavage domain and second DNA-cleavage domain generates a double- stranded break in a targeted DNA.

In some embodiments, the endonuclease domain comprises FokI or a portion thereof. In some embodiments, the endonuclease domain comprises a DNA-cleavage domain of FokI. In some embodiments, the endonuclease domain does not include a DNA-binding domain of FokI. FokI is a Type IIS restriction enzyme isolated from Flavobacterium okeanokoites . Each monomer of wildtype FokI has a DNA-binding domain and a DNA-cleavage domain. Wild-type FokI forms a dimer in which each monomer cleaves a single strand of DNA, leading to a double stranded break in the targeted DNA. See, e.g.. Wah et al. PNAS (1998) 95(18): 10564-10569. In some embodiments, the first DNA-cleavage domain and/or the second DNA-cleavage domain of the endonuclease domain is a DNA-cleavage domain of FokI or is derived from a DNA-cleavage domain of FokI. In some embodiments, the endonuclease domain does not comprise the DNA binding domain of FokI. In some embodiments, the endonuclease domain is not capable of forming and/or maintaining a complex with DNA in the absence of an accompanying Cpfl domain.

In some embodiments, the endonuclease domain is genetically modified relative to a naturally occurring or wildtype endonuclease domain sequence. In some embodiments, the first DNA-cleavage domain and/or the second DNA-cleavage domain comprise one or more modifications (e.g., mutations, substitutions, deletions, insertions) relative to a corresponding wildtype DNA-cleavage domain sequence. In some embodiments, the first DNA-cleavage domain and/or the second DNA-cleavage domain comprise one or more modifications to modulate activity of the endonuclease domain (or DNA-cleavage domain) such that at least one of the first DNA-cleavage domain or the second DNA-cleavage domain has reduced or eliminated endonuclease activity (e.g., does not cleave a phosphodiester bond). In some embodiments, the first DNA-cleavage domain comprises one or more modifications such that the first DNA-cleavage domain has reduced or eliminated endonuclease activity (e.g., does not cleave a phosphodiester bond). In some embodiments, the second DNA-cleavage domain comprises one or more modifications such that the second DNA-cleavage domain has reduced or eliminated endonuclease activity (e.g., does not cleave a phosphodiester bond).

In some embodiments, the first DNA-cleavage domain comprises one or more modifications such that the first DNA-cleavage domain has reduced or eliminated endonuclease activity (e.g., does not cleave a phosphodiester bond) and the second DNA- cleavage domain comprises wildtype or substantially wildtype endonuclease activity (e.g., functional endonuclease activity, capable of cleaving a phosphodiesterase bond), such that a dimer of the first DNA-cleavage domain and second DNA-cleavage domain does not produce double stranded breaks in a targeted DNA. In some embodiments, the first DNA-cleavage domain comprises one or more modifications such that the first DNA-cleavage domain has reduced or eliminated endonuclease activity (e.g., does not cleave a phosphodiester bond) and the second DNA-cleavage domain comprises wildtype or substantially wildtype endonuclease activity (e.g., functional endonuclease activity, capable of cleaving a phosphodiesterase bond), such that a dimer of the first DNA-cleavage domain and second DNA-cleavage domain is capable of generating a single-stranded break in a targeted DNA (e.g., is a nickase). In some embodiments, the second DNA-cleavage domain comprises one or more modifications such that the second DNA-cleavage domain has reduced or eliminated endonuclease activity (e.g., does not cleave a phosphodiester bond) and the first DNA- cleavage domain comprises wildtype or substantially wildtype endonuclease activity (e.g., functional endonuclease activity, capable of cleaving a phosphodiesterase bond), such that a dimer of the first DNA-cleavage domain and second DNA-cleavage domain does not produce double stranded breaks in a targeted DNA. In some embodiments, the second DNA-cleavage domain comprises one or more modifications such that the second DNA-cleavage domain has reduced or eliminated endonuclease activity (e.g.. does not cleave a phosphodiester bond) and the first DNA-cleavage domain comprises wildtype or substantially wildtype endonuclease activity (e.g.. functional endonuclease activity, capable of cleaving a phosphodiesterase bond), such that a dimer of the first DNA-cleavage domain and second DNA-cleavage domain is capable of generating a single-stranded break in a targeted DNA (e.g.. is a nickase).

In some embodiments, the first DNA-cleavage domain comprises one or more modifications that reduce or eliminate endonuclease activity of the first DNA-cleavage domain (e.g., does not cleave a phosphodiester bond). In some embodiments, the first DNA- cleavage domain comprises one or more mutations (e.g., 1, 2, 3, 4, 5 or more) that result in a DNA-cleavage domain having reduced or eliminated endonuclease activity. In some embodiments, the first DNA-cleavage domain comprises a mutation of one or more amino acids (e.g., 1, 2, 3, 4, 5 or more) that result in a DNA-cleavage domain having reduced or eliminated endonuclease activity. In some embodiments, the first DNA-cleavage domain comprises a mutation of one or more amino acids (e.g., 1, 2, 3, 4, 5 or more) in the catalytic site (active site) of the DNA-cleavage domain that result in a DNA-cleavage domain having reduced or eliminated endonuclease activity.

In some embodiments, the endonuclease domain comprises FokI or a portion thereof. In some embodiments, the endonuclease domain comprises a DNA-cleavage domain of FokI. In some embodiments, the endonuclease domain does not include a DNA-binding domain of FokI. In some embodiments, the endonuclease domain comprises a first DNA-cleavage domain from FokI. In some embodiments, the endonuclease domain comprises a second DNA-cleavage domain from FokI. In some embodiments, the first DNA-cleavage domain and/or the second DNA-cleavage domain from FokI comprises a mutation of one or more amino acids (e.g., 1, 2, 3, 4, 5 or more) that results in the DNA-cleavage domain having reduced or eliminated endonuclease activity, for example as compared to the wildtype DNA- cleavage domain from FokI (not comprising the mutation). Mutations in the DNA-cleavage domain to impair endonuclease activity of a monomer of a FokI dimer may direct DNA cleavage (nicking) to a particular DNA strand. See, e.g., Sanders et al. Nucleic Acids Res. (2009) 37(7): 2105-2115, incorporated by reference in its entirety. In some embodiments, the endonuclease domain comprises an amino acid sequence of SEQ ID NOs: 10-14, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity to SEQ ID NOs: 10-14. In some embodiments, the first DNA-cleavage domain and/or the second DNA-cleavage domain comprises an amino acid sequence of SEQ ID NOs: 10-14, or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity to SEQ ID NOs: 10-14.

In some embodiments, the first DNA-cleavage domain and/or the second DNA- cleavage domain comprises a DNA-cleavage domain from Fokl. In some embodiments, the first DNA-cleavage domain and/or the second DNA-cleavage domain comprises an amino acid sequence of SEQ ID NOs: 10-14 and comprises a substitution mutation of one or more amino acids (e.g., 1, 2, 3, 4, 5 or more), for example in the catalytic site (active site) of the DNA-cleavage domain, as compared to SEQ ID NO: 10 or 11, respectively, that results in a DNA-cleavage domain having reduced or eliminated endonuclease activity. In some embodiments, the first DNA-cleavage domain and/or the second DNA-cleavage domain comprises an amino acid sequence of SEQ ID NOs: 10 or 11 and comprises a substitution of an aspartic acid residue at amino acid position number 450 (which may also be referred to as D450) of SEQ ID NO: 10. In some embodiments, the first DNA-cleavage domain and/or the second DNA-cleavage domain comprises an amino acid sequence of SEQ ID NOs: 10 or 11 and comprises a substitution of an aspartic acid residue at amino acid position number 450 to an alanine (which may be referred to as D450A). In some embodiments, the first DNA- cleavage domain and/or the second DNA-cleavage domain comprises a substitution of an amino acid residue corresponding to the aspartic acid residue at amino acid position number 450 (which may be referred to as D450) of SEQ ID NO: 10. Exemplary Fokl and Fokl cleavage domain sequences are provided with the aspartic acid residue at position 450 is indicated in boldface with underline, in SEQ ID NO: 10 and 11 below.

Exemplary amino acid sequence of Fokl (SEQ ID NO: 10)

MVSKIRTFGWVQNPGKFENLKRWQVFDRNSKVHNEVKNIKIPTLVKESKIQKELVAI MNQHDLI YTYKELVGTG TS IRSEAPCDAI IQAT IADQGNKKGYIDNWS SDGFLRWAHALGF IEYINKSDSFVI TDVGLAYSKSADGSAIEKE ILIEAI S SYPPAIRILTLLEDGQHLTKFDLGKNLGFSGESGFTSLPEGILLDTLANAMPKDKGE IRNNWEGS SDK YARMIGGWLDKLGLVKQGKKEF I IPTLGKPDNKEF I SHAFKI TGEGLKVLRRAKGSTKFTRVPKRVYWEMLATNL TDKEYVRTRRALILE ILIKAGSLKIEQIQDNLKKLGFDEVIET IEND IKGLINTGIF IE IKGRFYQLKDHILQFV IPNRGVTKQLVKSELEEKKSELRHKLKYVPHEYIELIE IARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPD GAIYTVGSP IDYGVIVDTKAYSGGYNLP IGQADEMQRYVEENQTRNKHINPNEWWKVYP S SVTEFKFLFVSGHFK GNYKAQLTRLNHI TNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGE INF Exemplary amino acid sequence of FokI DNA cleavage domain (SEO ID NO: 11):

QLVKSELEEKKSELRHKLKYVPHEYIELIE IARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGS P IDYGVIVDTKAYSGGYNLP IGQADEMQRYVEENQTRNKHINPNEWWKVYP S SVTEFKFLFVSGHFKGNYKAQLT RLNHI TNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGE INF

Exemplary amino acid sequence of FokI DNA cleavage domain mutant (D450A) (SEO ID NO: 12)

QLVKSELEEKKSELRHKLKYVPHEYIELIE IARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPAGAIYTVGS P IDYGVIVDTKAYSGGYNLP IGQADEMQRYVEENQTRNKHINPNEWWKVYP S SVTEFKFLFVSGHFKGNYKAQLT RLNHI TNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGE INF

Exemplary amino acid sequence of an endonuclease domain comprising a FokI nickase (FokI DNA cleavage domain mutant (D450A) and FokI DNA cleavage domain separated by linker) (SEQ ID NO: 13). The first FokI DNA cleavage domain is shown in underline, a polypeptide linker is shown in italics, and a second FokI DNA cleavage domain is shown in boldface.

The D450A mutation is shown in the first FokI DNA cleavage domain in boldface with double underline.

QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRG KHLGGSRKPAG Al YTVGSP IDYGVIVDTKAYSGGYNLP IGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLF VSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF GSGSGSGS ITRT TNPRNWPKI YMSA GSIPLTTHI TNS IQP TL WTI GS INGVAPLAKS IKLGIP VTGSAYTD QTTA

MVRKKVSVFMGSGSGSGSSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQD RILEMKVMEFF MKVYGYRGKHLGGSRKPDGAI YTVGSP IDYGVIVDTKAYSGGYNLP IGQADEMQRYVEENQTRNKHIN PNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKA GTLTLEEV RRKFNNGEINF ( SEQ ID NO : 13 )

Exemplary amino acid sequence of an endonuclease domain comprising a FokI nickase (FokI DNA cleavage domain and FokI DNA cleavage domain mutant (D450A) separated by a linker) (SEQ ID NO: 14). The first FokI DNA cleavage domain is shown in underline, a polypeptide linker is shown in italics, and a second FokI DNA cleavage domain is shown in boldface. The D450A mutation is shown in the second FokI DNA cleavage domain in boldface with underline.

QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRG KHLGGSRKPDG Al YTVGSP IDYGVIVDTKAYSGGYNLP IGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLF VSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF GSGSGSGS ITRT TNPRNWPKI YMSA GSIPLTTHI TNS IQP TL WTI GS INGVAPLAKS IKLGIP VTGSAYTD QTTA MVRKKVSVFMGSGSGSGSSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRIL EMKVMEFF MKVYGYRGKHLGGSRKPAGAI YTVGSP IDYGVIVDTKAYSGGYNLP IGQADEMQRYVEENQTRNKHIN PNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKA GTLTLEEV RRKFNNGEINF ( SEQ ID NO : 14 ) Genomic Modification Domain

In some embodiments, the fusion polypeptides described herein comprise a Cpf 1 domain that lacks nuclease activity, an endonuclease domain, and a genomic modification domain. As used herein, a “genomic modification domain” refers to an enzyme, or portion thereof, that is capable of effecting a modification on the genome of a host cell. Examples of genomic modification domains, including epigenetic modifiers (e.g. a DNA methyltransferase, a DNA methylase, a histone acetyltransferase, a histone deacetylase, a histone methyltransferase, a histone methylase, or a functional portion or combination of any thereof) and enzyme that modify nucleic acids or polynucleotides, and/or act on nucleic acids or polynucleotides, such as helicases, polymerases, nucleases, ligases, transcription factors.

In some embodiments, the genomic modification domain comprises a base editor, which may refer to an enzyme or portion thereof that modifies a nucleobase of a polynucleotide. In some embodiments, the genomic modification domain comprises more than one base editor, or base editing domain. In some embodiments, the genomic modification domain comprises a deaminase enzyme, or portion thereof, which is capable of catalyzing a deamination reaction. In general, a deaminase, such as a cytosine or adenosine deaminase, target and deaminates a specific nucleobase, e.g., a cytosine or adenosine nucleobase of a C or A nucleotide. In methods of “base editing” deamination of a specific nucleobase, via cellular mismatch repair mechanisms, results in a change from a C to a T nucleotide, or a change from an A to a G nucleotide. See, e.g., Komor et al. Nature (2016) 533: 420-424; Rees et al. Nat. Rev. Genet. (2018) 19(12): 770-788; Anzalone et al. Nat. Biotechnol. (2020) 38: 824-844.

Base editors typically comprise a catalytically inactive Cas nuclease fused to a functional domain, e.g., a deaminase domain. See, e.g., Eid et al. Biochem. J. (2018) 475(11): 1955-1964; Rees et al. Nature Reviews Genetics (2018) 19:770-788. The fusion polypeptides described herein comprise Cpfl domain lacking nuclease activity, an endonuclease domain, and a genomic modification domain, which may be a base editing domain (e.g., a deaminase). In some embodiments, the fusion polypeptide comprises a cytidine deaminase, or portion thereof. Such fusion polypeptides may be referred to as cytosine base editors (CBE). In general, a cytidine deaminase catalyzes the hydrolysis of cytidine or deoxycytidine to uridine or deoxyuridine. In some embodiments, the cytidine deaminase catalyzes the hydrolysis of cytosine to uracil.

In some embodiments, the fusion polypeptide comprises an adenine deaminase, or portion thereof. Such fusion polypeptides may be referred to as adenine base editors (ABE). In general, an adenosine deaminase catalyzes the deamination of adenine in a deoxyadenosine residue. In some embodiments, the adenine deaminase catalyzes conversion of adenosine to inosine. In some embodiments, the adenine deaminase is a tRNA adenosine deaminase (TadA) or a variant thereof (e.g., an evolved variant such as TadA2.1).

In some embodiments, the fusion polypeptide comprises an adenine deaminase and a cytidine deaminase, or portions thereof. Such fusion polypeptides may be referred to as adenine and cytosine base editors.

In some embodiments, the fusion polypeptide comprises, from N-terminus to C- terminus, the Cas nuclease, the endonuclease domain, the adenine deaminase, and the cytidine deaminase. In some embodiments, the fusion polypeptide comprises, from N- terminus to C-terminus, the Cas nuclease, the endonuclease domain, the cytidine deaminase, and the adenine deaminase. In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, the Cas nuclease, the adenine deaminase, the endonuclease domain, and the cytidine deaminase. In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, the Cas nuclease, the adenine deaminase, the cytidine deaminase, and the endonuclease domain. In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, the Cas nuclease, the cytidine deaminase, the endonuclease domain, and the adenine deaminase. In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, the Cas nuclease, the cytidine deaminase, the adenine deaminase, and the endonuclease domain. In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, the endonuclease domain, the Cas nuclease, the adenine deaminase, and the cytidine deaminase. In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, the endonuclease domain, the Cas nuclease, the cytidine deaminase, and the adenine deaminase. In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, the endonuclease domain, the adenine deaminase, the Cas nuclease, and the cytidine deaminase. In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, the endonuclease domain, the adenine deaminase, the cytidine deaminase, and the Cas nuclease. In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, the endonuclease domain, the cytidine deaminase, the Cas nuclease, and the adenine deaminase. In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, the endonuclease domain, the cytidine deaminase, the adenine deaminase, and the Cas nuclease. In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, the adenine deaminase, the Cas nuclease, the endonuclease domain, and the cytidine deaminase. In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, the adenine deaminase, the Cas nuclease, the cytidine deaminase, and the endonuclease domain. In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, the adenine deaminase, the endonuclease domain, the Cas nuclease, and the cytidine deaminase. In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, the adenine deaminase, the endonuclease domain, the cytidine deaminase, and the Cas nuclease. In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, the adenine deaminase, the cytidine deaminase, the Cas nuclease, and the endonuclease domain. In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, the adenine deaminase, the cytidine deaminase, the endonuclease domain, and the Cas nuclease. In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, the cytidine deaminase, the Cas nuclease, the endonuclease domain, and the adenine deaminase. In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, the cytidine deaminase, the Cas nuclease, the adenine deaminase, and the endonuclease domain. In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, the cytidine deaminase, the endonuclease domain, the Cas nuclease, and the adenine deaminase. In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, the cytidine deaminase, the endonuclease domain, the adenine deaminase, and the Cas nuclease. In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, the cytidine deaminase, the adenine deaminase, the endonuclease domain, and the Cas nuclease. In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, the cytidine deaminase, the adenine deaminase, the Cas nuclease, and the endonuclease domain.

Cytidine deaminases and/or adenosine deaminases for use in the fusion polypeptides described herein may be obtained from any source known in the art. For example, in some embodiments, the cytidine deaminase and/or adenosine deaminase, or portion thereof, is from a naturally occurring deaminase or is a variant of a naturally occurring deaminase. In some embodiments, the cytidine deaminase and/or adenosine deaminase, or portion thereof, is an engineered or synthetic deaminase that is not naturally occurring.

Additional examples of suitable genomic modification domains for use in the fusion polypeptides described herein may be found, without limitation, in the exemplary base editors: BE1, BE2, BE3, HF-BE3, BE4, BE4max, AncBE4max, BE4-Gam, YE1-BE3, EEBES, YE2-BE3, YEE-CE3, VQR-BE3, VRER-BE3, SaBE3, SaBE4, SaBE4-Gam, Sa(KKH)- BE3, Target-AID, Target-AID-NG, AID, CDA1, APOBEC-1, APOBEC3G, xBE3, eA3A- BE3, BE-PLUS, TAM, CRISPR-X, ABE7.9, ABE7.10, ABE7.10*, xABE, AB ESa, VQR- ABE, VRER-ABE, Sa(KKH)-ABE, and CRISPR-SKIP. Additional examples of base editors can be found, for example, in U.S. Publication No. 2018/0312825A1, U.S. Publication No. 2018/0312828A1, and PCT Publication No. WO 2018/165629A1, which are incorporated by reference herein in their entireties. In some embodiments, the genomic modification is a cytosine deaminase, such as APOB EC (also referred to as “apolipoprotein B editing complex catalytic subunit 1,” APOBEC-1), pmCDAl, or activation-induced cytidine deaminase (AID). In some embodiments, the genomic modification is an adenine deaminase, such as TadA. In some embodiments, the endonuclease comprises an uracil glycosylase inhibitor (UGI). In some embodiments, the endonuclease comprises an adenine base editor (ABE), for example an ABE evolved from the RNA adenine deaminase TadA.

In some embodiments, the genomic modification domain comprises an amino acid sequence of SEQ ID NOs: 15, or a sequence with at least 80, 85, 90, 95, or 99% identity to any thereof.

Exemplary amino acid sequence of APOBEC-1 (SEQ ID NO: 15)

GSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQN TNKHVEVNFIE KFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNR QGLRDLIS SGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNIL RRKQPQLT FFTIALQSCHYQRLPPHILWATGLK

Linker Domains

Any of the fusion polypeptides described herein may further comprises one or more linker domains. A linker domain is an amino acid sequence by which two polypeptide domains may be joined. In general, a linker domain may be used, for example, to join adjacent domains or functional regions of a polypeptide and may allow a level of flexibility (or rigidity) such that the joined domains or regions are independently functional.

Exemplary linker domains are recited, for example, in Chen, et al, Adv Drug Deliv Rev (2013) Oct. 15 65(10): 1357-1369, however, one of skill in the art would not be limited by this disclosure. The linker may comprise any suitable amino acid sequence. In some embodiments, the linker domain is a flexible linker. Flexible linkers typically largely comprise small and/or polar amino acids, such as glycine (Gly) and serine (Ser) or threonine (Thr), respectively. This promotes flexibility and solubility in the resultant fusion polypeptide. Example flexible linker domains include, but are not limited to, glycine linkers (e.g., (Gly)s linkers) (SEQ ID NO: 54), serine linkers, glycine- serine linkers (e.g., (Gly-Gly- Gly-Ser)n (SEQ ID NO: 55) and (Gly-Gly-Gly-Gly-Ser) ₄ (SEQ ID NO: 56) linkers), and glycine-serine rich linkers (e.g., KESGSVSSEQLAQFRSLD (SEQ ID NO: 16), EGKSSGSGSESKST (SEQ ID NO: 17), and GSAGSAAGSGEF(SEQ ID NO: 18)).

In some embodiments, the linker domain is a Gly/Ser linker from about 1 to about 100, from about 3 to about 20, from about 5 to about 30, from about 5 to about 18, or from about 3 to about 8 amino acids in length and consists of glycine and/or serine residues in sequence. Accordingly, the Gly/Ser linker may consist of glycine and/or serine residues. Preferably, the Gly/Ser linker comprises the amino acid sequence of GGGGS (SEQ ID NO: 19), and multiple SEQ ID NO: 19 may be present within the linker. Any linker sequence may be used as a spacer between any two domains or functional regions of any of the fusion polypeptides described herein, such as between the Cpfl domain and the endonuclease domain, and/or between a first DNA-cleavage domain and a second DNA-cleavage domain. In some, embodiments, the region linker is ([G] _x [S] _y ) _z (SEQ ID NO: 57), for example wherein x can be 1-10, 7 can be 1-3, and z can be 1-5. In some embodiments, the linker region comprises the amino acid sequence GGGGSGGGGS (SEQ ID NO: 20). In some embodiments, the linker region comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 21).

In some embodiments, the linker is an XTEN linker, which is an unstructured polypeptide consisting of hydrophilic residues of varying lengths. Amino acid sequences of XTEN peptides will be evident to one of skill in the art and can be found, for example, in U.S. Pat. No. 8,673,860, which is herein incorporated by reference. In some embodiments, the XTEN linker is provided by SEQ ID NO: 22.

Amino acid sequence of XTEN linker (SEQ ID NO: 22) SGSETPGTSESATPES

Amino acid sequence of exemplary linker domain (SEQ ID NO: 23) GSGSGSGS I TRTTNPRNWPKIYMSAGS IPLTTHI TNS IQPTLWT IGS INGVAPLAKS IKLGIPVTGSAYTDQTT AMVRKKVSVFMGSGSGSGS S

In some embodiments, the linker domain is a rigid linker. Non-limiting examples of rigid linkers are known in the art and can be found, for example, in Tan, el al. Nat. Commun. (2019) 10: 439. Rigid linkers often include proline (Pro) residues, which contribute to rigidity of a protein sequence because the contain a secondary amine. Fusion

The domains described herein may be arranged in any order (from N-terminus to C- terminus) in a fusion polypeptides described herein, such that each of the domains is capable of performing its respective function.

In some embodiments, a fusion polypeptide described herein may comprise a Cpf 1 domain that is located at the N-terminus of the endonuclease domain. In some embodiments, the endonuclease domain comprises a DNA-cleavage domain and the Cpfl domain that is located N-terminal of the DNA-cleavage domain. In some embodiments, the endonuclease domain comprises a first DNA-cleavage domain and a second DNA-cleavage domain, and the Cpfl domain that is located N-terminal of both the first and second DNA-cleavage domains.

In some embodiments, a fusion polypeptide described herein may comprise an endonuclease domain that is located at the N-terminus of the Cpfl domain. In some embodiments, the endonuclease domain comprises a DNA-cleavage domain, and the DNA- cleavage domain that is located N-terminal of the Cpfl domain. In some embodiments, the endonuclease domain comprises a first DNA-cleavage domain and a second DNA-cleavage domain, and both the first and second DNA-cleavage domain are located N-terminal of the Cpfl domain.

Any of the fusion polypeptides described herein may further comprise a genomic modification domain. In some embodiments, a fusion polypeptide described herein may comprise a genomic modification domain that is located N-terminal of the Cpfl domain. In some embodiments, a fusion polypeptide described herein may comprise a genomic modification domain that is located N-terminal of the endonuclease domain. In some embodiments, a fusion polypeptide described herein may comprise a genomic modification that is located between the Cpfl domain and the endonuclease domain.

In some embodiments, the fusion polypeptide comprises, from N-terminus to C- terminus, the Cpfl domain, the endonuclease domain, and the genomic modification domain. In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, the Cpfl domain, the genomic modification domain, and the endonuclease domain. In some embodiments, the fusion polypeptide comprises from, N-terminus to C-terminus, the endonuclease domain, the Cpfl domain, and the genomic modification domain. In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, the endonuclease domain, the genomic modification domain, and the Cpfl domain. In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, the genomic modification domain, the Cpfl domain, and the endonuclease domain. In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, the genomic modification domain, the endonuclease domain, and the Cpfl domain.

In some embodiments, the fusion polypeptide comprises, from N-terminus to C- terminus, the Cpfl domain comprising any of SEQ ID NOs: 3 or 5, the endonuclease domain, and the genomic modification domain. In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, the Cpfl domain comprising any of SEQ ID NOs: 3 or 5, the genomic modification domain, and the endonuclease domain. In some embodiments, the fusion polypeptide comprises from, N-terminus to C-terminus, the endonuclease domain, the Cpfl domain comprising any of SEQ ID NOs: 3 or 5, and the genomic modification domain. In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, the endonuclease domain, the genomic modification domain, and the Cpfl domain comprising any of SEQ ID NOs: 3 or 5. In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, the genomic modification domain, the Cpfl domain comprising any of SEQ ID NOs: 3 or 5, and the endonuclease domain. In some embodiments, the fusion polypeptide comprises, from N-terminus to C- terminus, the genomic modification domain, the endonuclease domain, and the Cpfl domain comprising any of SEQ ID NOs: 3 or 5.

In some embodiments, the fusion polypeptide comprises, from N-terminus to C- terminus, the Cpfl domain, the endonuclease domain, and the deamination domain. In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, the Cpfl domain, a deamination domain, and the endonuclease domain. In some embodiments, the fusion polypeptide comprises from, N-terminus to C-terminus, the endonuclease domain, the Cpfl domain, and the deamination domain. In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, the endonuclease domain, the deamination domain, and the Cpfl domain. In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, a deamination domain, the Cpfl domain, and the endonuclease domain. In some embodiments, the fusion polypeptide comprises, from N- terminus to C-terminus, the deamination domain, the endonuclease domain, and the Cpfl domain.

In some embodiments, the fusion polypeptide comprises, from N-terminus to C- terminus, the Cpfl domain comprising any of SEQ ID NOs: 3 or 5, the endonuclease domain, and the deamination domain. In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, the Cpfl domain comprising any of SEQ ID NOs: 3 or 5, the deamination domain, and the endonuclease domain. In some embodiments, the fusion polypeptide comprises from, N-terminus to C-terminus, the endonuclease domain, the Cpfl domain comprising any of SEQ ID NOs: 3 or 5, and the deamination domain. In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, the endonuclease domain, the deamination domain, and the Cpfl domain comprising any of SEQ ID NOs: 3 or 5. In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, the deamination domain, the Cpfl domain comprising any of SEQ ID NOs: 3 or 5, and the endonuclease domain. In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus, the deamination domain, the endonuclease domain, and the Cpfl domain comprising any of SEQ ID NOs: 3 or 5.

Any of the fusion polypeptides described herein may further comprise one or more linker domains. In some embodiments, the fusion polypeptide comprises a linker domain between the Cpfl domain and the endonuclease domain. In some embodiments, the fusion polypeptide comprises a linker domain between the Cpfl domain and the genomic modification domain. In some embodiments, the fusion polypeptide comprises a linker domain between the endonuclease domain and the genomic modification domain.

In some embodiments, the endonuclease domain comprises a linker domain between a first DNA-cleavage domain and a second DNA-cleavage domain.

Amino acid sequences of exemplary fusion polypeptides of the present disclosure are provided below.

1. Construct A

An exemplary fusion polypeptide, as described herein, comprises a first FokI DNA cleavage domain, a polypeptide linker, a second FokI DNA cleavage domain comprising a D450A mutation, an XTEN linker, and a Cpfl domain that lacks nuclease activity.

In some embodiments, the fusion polypeptide comprises an amino acid sequence shown in SEQ ID NO: 24, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 24. In SEQ ID NO: 24 below, the first FokI DNA cleavage domain is shown in underline, the polypeptide linker is shown in italics, the second FokI DNA cleavage domain containing an D450A mutation is shown in underline (with mutation indicated in boldface), the XTEN linker shown in italics, and the AsCpfl lacking nuclease activity is shown in boldface. ‘Fokll’, as used in sequence descriptions herein, refers to the second FokI DNA cleavage domain in an exemplary construct (from N to C), regardless of the presence or absence of a mutation in the first or second FokI DNA cleavage domains.

Amino acid sequence of Construct A: FokI (D450) - polypeptide linker - Fokll (D450A) -

XTEN linker - AsCpfl (A) (SEQ ID NO: 24)

MQLVKSELEEKKSELRHKLKYVPHEYIELIE IARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVG SP IDYGVIVDTKAYSGGYNLP IGQADEMQRYVEENQTRNKHINPNEWWKVYP S SVTEFKFLFVSGHFKGNYKAQL TRLNHI TNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGE INFGSGSGSGST TR TTNPRNWPKI YMSAGS IPLTTHITNSIQPTLWTIGSINGVAPLAKSIKLGIPVTGSAYTDQTTAMVRKKVSVFMGS GSGSGSSQIAVKSE'LIE EKKSELRHKLKYVPHEYIELIE IARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPAGAIYTVGSP IDYGVIV DTKAYSGGYNLP IGQADEMQRYVEENQTRNKHINPNEWWKVYP S SVTEFKFLFVSGHFKGNYKAQLTRLNHI TNC NGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGE INFSGSETPGTSESATPESTQFEGFTNLYQVSKTLRFELI PQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDS YRKEKTEETRNALIE EQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENA LLRSFDKFTTYFSGF YRNRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIF VSTSIEEVFSFPFYN QLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFK QILSDRNTLSFILEE FKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHW DTLRNALYERRISEL TGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTT LKKQEEKEILKSQLD SLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFK LNFQMPTLARGWDVN VEKNRGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPK CSTQLKAVTAHFQTH TTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSS LRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGK PNLHTLYWTGLFSPE NLAKTS IKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTP IPDTLYQELYDYVNHRLSHDLSDEARALLP NVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGI ARGERNLIYITVIDS TGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSWGTIKDLKQGYLSQVIHEIVDLM IHYQAWVLENLNF GFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGT QSGFLFYVPAPYTSK IDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGF MPAWDIVFEKNETQF DAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLEND DSHAIDTMVALIRSV LQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLK ESKDLKLQNGISNQD WLAYIQELRN

2. Construct B

An exemplary fusion polypeptide, as described herein, comprises an APOB EC- 1 base editor, a Cpfl domain that lacks nuclease activity, an XTEN linker, a first FokI DNA cleavage domain comprising a D450A mutation, a polypeptide linker, and a second FokI DNA cleavage domain.

In some embodiments, the fusion polypeptide comprises an amino acid sequence shown in SEQ ID NO: 25, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 25. In SEQ ID NO: 25 below, the APOBEC-1 base editor is shown in underline, a linker sequence is shown in italics, the AsCpfl lacking nuclease activity is shown in boldface, the XTEN linker shown in italics, first FokI DNA cleavage domain containing an D450A mutation is shown in underline (with mutation indicated in boldface), the polypeptide linker is shown in italics, the second FokI DNA cleavage domain is shown in underline.

Amino acid sequence of Construct B: APOBEC Base Editor - AsCpfl (D908A) - XTEN -

FokI (D450A) - polypeptide linker - Fokll (D450) (SEQ ID NO: 25)

GS SETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYE INWGGRHS IWRHTSQNTNKHVEVNF IEKFTTERY FCPNTRCS I TWFLSWSPCGECSRAI TEFLSRYPHVTLF IYIARLYHHADPRNRQGLRDLI S SGVT IQIMTEQESG YCWRNFVNYSP SNEAHWPRYPHLWVRLYVLELYC I ILGLPPCLNILRRKQPQLTFFT IALQSCHYQRLPPHILWA TGLKSGGSSGGSSGSETPGTSESATPESSGGSSGGSTQFEGFTNLYQVSKTLRFELIPQG KTLKHIQEQGFIEED KARNDHYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQA TYRNAIHDYFIGRTD NLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYRN RKNVFSAEDISTAIP HRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLL TQTQIDLYNQLLGGI SREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKS DEEVIQSFCKYKTLL RNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGK ITKSAKEKVQRSLKH EDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLL GLYHLLDWFAVDESN EVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLARGWDVNVEK NRGAILFVKNGLYYL GIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTP ILLSNNFIEPLEITK EIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRP SSQYKDLGEYYAELN PLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLA KTSIKLNGQAELFYR PKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVI TKEVSHEIIKDRRFT SDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIARGERNLIYITVIDSTGK ILEQRSLNTIQQFDY QKKLDNREKERVAARQAWSWGTIKDLKQGYLSQVIHEIVDLMIHYQAWVLENLNFGFKSK RTGIAEKAVYQQF EKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDP LTGFVDPFVWKTIKN HESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAK GTPFIAGKRIVPVIE NHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQM RNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLA YIQELRNSGSETPGT SESATPESQLVKSELEEKKSELRHKLKYVPHEYIELIE IARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPA GAIYTVGSP IDYGVIVDTKAYSGGYNLP IGQADEMQRYVEENQTRNKHINPNEWWKVYP S SVTEFKFLFVSGHFK GNYKAQLTRLNHI TNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGE INFGSGSGSGSTTPTTNPRNWPJC IYMSAGSIPLTTHITNSIQPTLWTIGSINGVAPLAKSIKLGIPVTGSAYTDQTTAMVRKK VSVFMGSGSGSGSSQ LVKSELEEKKSELRHKLKYVPHEYIELIE IARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSP IDYGVIVDTKAYSGGYNLP IGQADEMQRYVEENQTRNKHINPNEWWKVYP S SVTEFKFLFVSGHFKGNYKAQLTR LNH I TNCNGAVLS VEELL I GGEMI KAGTLTLEEVRRKFNNGE INF

3. Construct C

An exemplary fusion polypeptide, as described herein, comprises an APOBEC- 1 base editor, a MAD7™-based domain that lacks nuclease activity, an XTEN linker, a first FokI DNA cleavage domain comprising a D450A mutation, a polypeptide linker, and a second FokI DNA cleavage domain.

In some embodiments, the fusion polypeptide comprises an amino acid sequence shown in SEQ ID NO: 26, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 26. In SEQ ID NO: 26 below, the APOBEC- 1 base editor is shown in underline, a linker sequence is shown in italics, the Mad7™-based domain lacking nuclease activity is shown in boldface, the XTEN linker shown in italics, first FokI DNA cleavage domain containing an D450A mutation is shown in underline (with mutation indicated in boldface), the polypeptide linker is shown in italics, the second FokI DNA cleavage domain is shown in underline.

Amino acid sequence of Construct C: APOBEC Base Editor - MAD7™(D908A) - XTEN -

FokI (D450A) - polypeptide linker - Fokll (D450) (SEQ ID NO: 26)

GS SETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYE INWGGRHS IWRHTSQNTNKHVEVNF IEKFTTERY FCPNTRCS I TWFLSWSPCGECSRAI TEFLSRYPHVTLF IYIARLYHHADPRNRQGLRDLI S SGVT IQIMTEQESG YCWRNFVNYSP SNEAHWPRYPHLWVRLYVLELYC I ILGLPPCLNILRRKQPQLTFFT IALQSCHYQRLPPHILWA TGLKSGGSSGGSSGSETPGTSE'SATPE'SSGGSSGGSNNGTNNFQNFIGISSLQKTLRN ALIPTETTQQFIVKNGI IKEDELRGENRQILKDIMDDYYRGFISETLSSIDDIDWTSLFEKMEIQLKNGDNKDTLIK EQTEYRKAIHKKFAN DDRFKNMFSAKLISDILPEFVIHNNNYSASEKEEKTQVIKLFSRFATSFKDYFRNRANCF SADDISSSSCHRIVN DNAEIFFSNALVYRRIVKSLSNDDINKISGDMKDSLKEMSLEEIYSYEKYGEFITQEGIS FYNDICGKVNSFMNL YCQKNKENKNLYKLQKLHKQILCIADTSYEVPYKFESDEEVYQSVNGFLDNISSKHIVER LRKIGDNYNGYNLDK IYIVSKFYESVSQKTYRDWETINTALEIHYNNILPGNGKSKADKVKKAVKNDLQKSITEI NELVSNYKLCSDDNI KAETYIHEISHILNNFEAQELKYNPEIHLVESELKASELKNVLDVIMNAFHWCSVFMTEE LVDKDNNFYAELEEI YDEIYPVISLYNLVRNYVTQKPYSTKKIKLNFGIPTLARGWSKSVEYSRNAIILMRDNLY YLGIFNAKNKPDKKI IEGNTSENKGDYKKMIYNLLPGPNKMIPKVFLSSKTGVETYKPSAYILEGYKQNKHIKSS KDFDITFCHDLIDYF KNCIAIHPEWKNFGFDFSDTSTYEDISGFYREVELQGYKIDWTYISEKDIDLLQEKGQLY LFQIYNKDFSKKSTG NDNLHTMYLKNLFSEENLKDIVLKLNGEAEIFFRKSSIKNPIIHKKGSILVNRTYEAEEK DQFGNIQIVRKNIPE NIYQELYKYFNDKSDKELSDEAAKLKNWGHHEAATNIVKDYRYTYDKYFLHMPITINFKA NKTGFINDRILQYI AKEKDLHVIGIARGERNLIYVSVIDTCGNIVEQKSFNIVNGYDYQIKLKQQEGARQIARK EWKEIGKIKEIKEGY LSLVIHEISKMVIKYNAIIAMEDLSYGFKKGRFKVERQVYQKFETMLINKLNYLVFKDIS ITENGGLLKGYQLTY IPDKLKNVGHQCGCIFYVPAAYTSKIDPTTGFVNIFKFKDLTVDAKREFIKKFDSIRYDS EKNLFCFTFDYNNFI TQNTVMSKS SWS VYT YGVRIKRRFVNGRFSNESDT ID I TKDMEKTLEMTD INWRDGHDLRQD I ID YE I VQHIFE I FRLTVQMRNSLSELEDRDYDRLISPVLNENNIFYDSAKAGDALPKDADANGAYCIALKGL YEIKQITENWKEDGK FSRDKLKISNKDWFDFIQNKRYLSGSETPGTSESATPESQLVKSELEEKKSELRHKLKYV PHEYIELIE IARNST QDRILEMKVMEFFMKVYGYRGKHLGGSRKPAGAIYTVGSP IDYGVIVDTKAYSGGYNLP IGQADEMQRYVEENQT RNKHINPNEWWKVYP S SVTEFKFLFVSGHFKGNYKAQLTRLNHI TNCNGAVLSVEELLIGGEMIKAGTLTLEEVR RKFNNGE INFGSGSGSGSITRTTNPRNWPKIYMSAGSIPLTTHITNSIQPTLWTIGSINGVAPLAKS IKLGIPV TGSAYTDQTTAWPKKVSVFMGSGSGSGSSQLVKSELEEKKSELRHKLKYVPHEYIELIE IARNSTQDRILEMKV MEFFMKVYGYRGKHLGGSRKPDGAIYTVGSP IDYGVIVDTKAYSGGYNLP IGQADEMQRYVEENQTRNKHINPNE WWKVYP S SVTEFKFLFVSGHFKGNYKAQLTRLNHI TNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGE IN F

4. Construct E

An exemplary fusion polypeptide, as described herein, comprises a Cpf 1 domain that lacks nuclease activity, an XTEN linker, a first FokI DNA cleavage domain, a polypeptide linker, and a second FokI DNA cleavage domain.

In some embodiments, the fusion polypeptide comprises an amino acid sequence shown in SEQ ID NO: 27, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 27. In SEQ ID NO: 27 below, the Cpfl domain lacking nuclease activity is shown in boldface, the XTEN linker shown in italics, first FokI DNA cleavage domain is shown in underline, the polypeptide linker is shown in italics, the second FokI DNA cleavage domain is shown in underline.

Amino acid sequence of Construct E: Cpf 1 (D908A) -XTEN - FokI- polypeptide linker -

Fokll (SEQ ID NO: 27)

TQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIY KTYADQCLQLVQLDWENL SAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAE LFNGKVLKQLGTVTT TEHENALLRSFDKFTTYFSGFYRNRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRL ITAVPSLREHFENVK KAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQ KNDETAHIIASLPHR FIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDL THIFISHKKLETISS ALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFK QKTSEILSHAHAALD QPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSL SFYNKARNYATKKPY SVEKFKLNFQMPTLARGWDVNVEKNRGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKT SEGFDKMYYDYFPDA AKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAK KTGDQKGYREALCKW IDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVE TGKLYLFQIYNKDFA KGHHGKPNLHTLYWTGLFSPENLAKTS IKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTP IPDTLYQEL YDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPS KFNQRVNAYLKEHPE TPIIGIARGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSWG TIKDLKQGYLSQVI HEIVDLMIHYQAWVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVG GVLNPYQLTDQFTS FAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTG DFILHFKMNRNLSFQ RGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALL EEKGIVFRDGSNILP KLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMD ADANGAYHIALKGQL LLNHLKESKDLKLQNGISNQDWLAYIQELRNSGSETPGTSESATPESQLVKSELEEKKSE LRHKLKYVPHEYIEL IE IARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSP IDYGVIVDTKAYSGGYNLP IGQADEMQ RYVEENQTRNKHINPNEWWKVYP S SVTEFKFLFVSGHFKGNYKAQLTRLNHI TNCNGAVLSVEELLIGGEMIKAG TLTLEEVRRKFNNGE INFGSGSGSGS I TRTTNPRNWPKIYMSAGS IPLTTHI TNS IQPTLWT IGS INGVAPLAK S IKLGIPVTGSAYTDQTTAMVRKKVSVFMGSGSGSGS SQLVKSELEEKKSELRHKLKYVPHEYIELIE IARNSTQ DRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSP IDYGVIVDTKAYSGGYNLP IGQADEMQRYVEENQTR NKHINPNEWWKVYP S SVTEFKFLFVSGHFKGNYKAQLTRLNHI TNCNGAVLSVEELLIGGEMIKAGTLTLEEVRR KFNNGE INF

5. Construct F

An exemplary fusion polypeptide, as described herein, comprises a first FokI DNA cleavage domain, a polypeptide linker, and a second FokI DNA cleavage domain, an XTEN linker, and a Cpfl domain that lacks nuclease activity.

In some embodiments, the fusion polypeptide comprises an amino acid sequence shown in SEQ ID NO: 28, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 28. In SEQ ID NO: 28 below, the first FokI DNA cleavage domain is shown in underline, the polypeptide linker is shown in italics, the second FokI DNA cleavage domain is shown in underline, the XTEN linker shown in italics, and the Cpfl domain lacking nuclease activity is shown in boldface. Amino acid sequence of Construct F: FokI- polypeptide linker - Fokll - XTEN - Cpfl (D908A) (SEQ ID NO: 28)

QLVKSELEEKKSELRHKLKYVPHEYIELIE IARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGS P IDYGVIVDTKAYSGGYNLP IGQADEMQRYVEENQTRNKHINPNEWWKVYP S SVTEFKFLFVSGHFKGNYKAQLT RLNHI TNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGE INFGSGSGSGSTTRTTNPRNWPJCTYMSAGST PL TTHITNSIQPTLWTIGSINGVAPLAKSIKLGIPVTGSAYTDQTTAMVRKKVSVFMGSGSG SGSSQIAVKSE'LIEE KKSELRHKLKYVPHEYIELIE IARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSP IDYGVIVD TKAYSGGYNLP IGQADEMQRYVEENQTRNKHINPNEWWKVYP S SVTEFKFLFVSGHFKGNYKAQLTRLNHI TNCN GAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGE INFSGSETPGTSESATPESTQFEGFTNLYQVSKTLRFELIP QGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSY RKEKTEETRNALIEE QATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENAL LRSFDKFTTYFSGFY RNRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFV STSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQ ILSDRNTLSFILEEF KSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWD TLRNALYERRISELT GKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTL KKQEEKEILKSQLDS LLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKL NFQMPTLARGWDVNV EKNRGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKC STQLKAVTAHFQTHT TPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDF LSKYTKTTSIDLSSL RPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKP NLHTLYWTGLFSPEN LAKTS IKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTP IPDTLYQELYDYVNHRLSHDLSDEARALLPN VITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIA RGERNLIYITVIDST GKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSWGTIKDLKQGYLSQVIHEIVDLMI HYQAWVLENLNFG FKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQ SGFLFYVPAPYTSKI DPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFM PAWDIVFEKNETQFD AKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDD SHAIDTMVALIRSVL QMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKE SKDLKLQNGISNQDW LAYIQELRN

6. Construct G

An exemplary fusion polypeptide, as described herein, comprises a Cpfl domain that lacks nuclease activity, an XTEN linker, a first FokI DNA cleavage domain (D450A), a polypeptide linker, and a second FokI DNA cleavage domain.

In some embodiments, the fusion polypeptide comprises an amino acid sequence shown in SEQ ID NO: 29, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 29. In SEQ ID NO: 29 below, the Cpfl domain lacking nuclease activity is shown in boldface, the XTEN linker shown in italics, first FokI DNA cleavage domain containing an D450A mutation is shown in underline (with mutation indicated in boldface), the polypeptide linker is shown in italics, and the second FokI DNA cleavage domain is shown in underline.

Amino acid sequence of Construct G: Cpfl (D908A) - XTEN - FokI (D450A)- polypeptide linker - Fokll (SEQ ID NO: 29) TQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTY ADQCLQLVQLDWENL SAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAE LFNGKVLKQLGTVTT TEHENALLRSFDKFTTYFSGFYRNRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRL ITAVPSLREHFENVK KAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQ KNDETAHIIASLPHR FIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDL THIFISHKKLETISS ALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFK QKTSEILSHAHAALD QPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSL SFYNKARNYATKKPY SVEKFKLNFQMPTLARGWDVNVEKNRGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKT SEGFDKMYYDYFPDA AKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAK KTGDQKGYREALCKW IDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVE TGKLYLFQIYNKDFA KGHHGKPNLHTLYWTGLFSPENLAKTS IKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTP IPDTLYQEL YDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPS KFNQRVNAYLKEHPE TPIIGIARGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSWG TIKDLKQGYLSQVI HEIVDLMIHYQAWVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVG GVLNPYQLTDQFTS FAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTG DFILHFKMNRNLSFQ RGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALL EEKGIVFRDGSNILP KLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMD ADANGAYHIALKGQL LLNHLKESKDLKLQNGISNQDWLAYIQELRNS GS E TP GT S E S ATP E SQLVKSELEEKKSELRHKLKYVPHEYIEL IE IARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPAGAIYTVGSP IDYGVIVDTKAYSGGYNLP IGQADEMQ RYVEENQTRNKHINPNEWWKVYP S SVTEFKFLFVSGHFKGNYKAQLTRLNHI TNCNGAVLSVEELLIGGEMIKAG TLTLEEVRRKFNNGE INF GSGSGSGSI TR TTNPRNWPKI YMSAGSIPL TTHI TNSIQP TL WTIGSINGVAPLAK STJCLGTP VTGSAYTDQTTAMVRJCJCVSVFMGSGSGSGSSQLVKSELEEKKSELRHKLKYVPHEYIE LIE I ARNSTQ DRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSP IDYGVIVDTKAYSGGYNLP IGQADEMQRYVEENQTR NKHINPNEWWKVYP S SVTEFKFLFVSGHFKGNYKAQLTRLNHI TNCNGAVLSVEELLIGGEMIKAGTLTLEEVRR KFNNGE INF

7. Construct H

An exemplary fusion polypeptide, as described herein, comprises a Cpf 1 domain that lacks nuclease activity, an XTEN linker, a first FokI DNA cleavage domain, a polypeptide linker, and a second FokI DNA cleavage domain (D450A).

In some embodiments, the fusion polypeptide comprises an amino acid sequence shown in SEQ ID NO: 30, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 30. In SEQ ID NO: 30 below, the Cpfl domain lacking nuclease activity is shown in boldface, the XTEN linker shown in italics, first FokI DNA cleavage domain is shown in underline, the polypeptide linker is shown in italics, and the second FokI DNA cleavage domain containing an D450A mutation is shown in underline (with mutation indicated in boldface).

Amino acid sequence of Construct H: Cpfl (D908A) - XTEN - FokI- polypeptide linker - Fokll (D450A) (SEQ ID NO: 30)

TQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIY KTYADQCLQLVQLDWENL SAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAE LFNGKVLKQLGTVTT TEHENALLRSFDKFTTYFSGFYRNRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRL ITAVPSLREHFENVK KAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQ KNDETAHIIASLPHR FIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDL THIFISHKKLETISS ALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFK QKTSEILSHAHAALD QPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSL SFYNKARNYATKKPY SVEKFKLNFQMPTLARGWDVNVEKNRGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKT SEGFDKMYYDYFPDA AKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAK KTGDQKGYREALCKW IDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVE TGKLYLFQIYNKDFA KGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKL KDQKTPIPDTLYQEL YDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPS KFNQRVNAYLKEHPE TPIIGIARGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSWG TIKDLKQGYLSQVI HEIVDLMIHYQAWVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVG GVLNPYQLTDQFTS FAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTG DFILHFKMNRNLSFQ RGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALL EEKGIVFRDGSNILP KLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMD ADANGAYHIALKGQL LLNHLKESKDLKLQNGISNQDWLAYIQELRNSGSETPGTSESATPESQLVKSELEEKKSE LRHKLKYVPHEYIEL IEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAY SGGYNLPIGQADEMQ RYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVL SVEELLIGGEMIKAG TLTLEEVRRKFNNGEINFGSGSGSGSITRTTNPRNW PKIYMSAGSIPLTTHITNSIQPTLWTIGSINGVAPLAK SIKLGIPVTGSAYTDQTTAMVRKKVSVFMGSGSGSGSSQLVKSELEEKKSELRHKLKYVP HEYIELIEIARNSTQ DRILEMKVMEFFMKVYGYRGKHLGGSRKPAGAIYTVGSP IDYGVIVDTKAYSGGYNLP IGQADEMQRYVEENQTR NKHINPNEWWKVYP S SVTEFKFLFVSGHFKGNYKAQLTRLNHI TNCNGAVLSVEELLIGGEMIKAGTLTLEEVRR KFNNGE INF

8. Construct I

An exemplary fusion polypeptide, as described herein, comprises a first FokI DNA cleavage domain (D450A), a polypeptide linker, a second FokI DNA cleavage domain, an XTEN linker, and a Cpfl domain that lacks nuclease activity.

In some embodiments, the fusion polypeptide comprises an amino acid sequence shown in SEQ ID NO: 31, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 31. In SEQ ID NO: 31 below, the first FokI DNA cleavage domain containing an D450A mutation is shown in underline (with mutation indicated in boldface), the polypeptide linker is shown in italics, the second FokI DNA cleavage domain, the XTEN linker shown in italics, and the Cpfl domain lacking nuclease activity is shown in boldface.

Amino acid sequence of Construct I: FokI (D450A) - polypeptide linker - Fokll - XTEN -

Cpfl (D908A) (SEQ ID NO: 31)

MQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYR GKHLGGSRKPAGAIYTVG SPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKF LFVSGHFKGNYKAQL TRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFGSGSGSGSITRTT NPRNW PKIYMSAGS IPLTTHITNSIQPTLWTIGSINGVAPLAKSIKLGIPVTGSAYTDQTTAMVRKKVSVFMGS GSGSGSSQUAKSKLE EKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDG AIYTVGSPIDYGVIV DTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKG NYKAQLTRLNHITNC NGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFSGSETPGTSESATPESTQFEGF TNLYQVSKTLRFELI PQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDS YRKEKTEETRNALIE EQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENA LLRSFDKFTTYFSGF YRNRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIF VSTSIEEVFSFPFYN QLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFK QILSDRNTLSFILEE FKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHW DTLRNALYERRISEL TGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTT LKKQEEKEILKSQLD SLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFK LNFQMPTLARGWDVN VEKNRGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPK CSTQLKAVTAHFQTH TTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSS LRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGK PNLHTLYWTGLFSPE NLAKTS IKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTP IPDTLYQELYDYVNHRLSHDLSDEARALLP NVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGI ARGERNLIYITVIDS TGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSWGTIKDLKQGYLSQVIHEIVDLM IHYQAWVLENLNF GFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGT QSGFLFYVPAPYTSK IDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGF MPAWDIVFEKNETQF DAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLEND DSHAIDTMVALIRSV LQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLK ESKDLKLQNGISNQD WLAYIQELRN

Nucleic Acids and Vectors

Also provided herein are nucleic acids comprising a nucleotide sequence encoding any of the fusion polypeptides described herein. In some embodiments, any nucleotide sequences herein may be codon-optimized. Without being bound to a particular theory or mechanism, it is believed that codon optimization of the nucleotide sequence increases the translation efficiency of the mRNA transcripts. Codon optimization of the nucleotide sequence may involve substituting a native codon for another codon that encodes the same amino acid, but can be translated by tRNA that is more readily available within a cell, thus increasing translation efficiency. Optimization of the nucleotide sequence may also reduce secondary mRNA structures that would interfere with translation, thus increasing translation efficiency. In an embodiment of the invention, the codon-optimized nucleotide sequence may comprise, consist, or consist essentially of any one of the nucleic acid sequences described herein.

Any of the nucleic acids of described herein may be recombinant. As used herein, the term “recombinant” refers to (i) molecules that are constructed outside living cells by joining natural or synthetic nucleic acid segments to nucleic acid molecules that can replicate in a living cell, or (ii) molecules that result from the replication of those described in (i) above. For purposes herein, the replication can be in vitro replication or in vivo replication.

A recombinant nucleic acid may be one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques, such as those described in Green et al., supra. The nucleic acids can be constructed based on chemical synthesis and/or enzymatic ligation reactions using procedures known in the art. See, for example, Green et al., supra. For example, a nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed upon hybridization (e.g., phosphorothioate derivatives and acridine substituted nucleotides). Examples of modified nucleotides that can be used to generate the nucleic acids include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5- chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5 -(carboxy hydroxy methyl) uracil, 5 -carboxy methylaminomethyl- 2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1 -methyl guanine, 1- methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5- methylcytosine, N6-substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5- methoxy aminomethyl-2- thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2- methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4- thiouracil, 5-methyluracil, uracil- 5 -oxy acetic acid methylester, 3-(3-amino-3-N-2- carboxypropyl) uracil, and 2,6- diaminopurine. Alternatively, one or more of the nucleic acids of the invention can be purchased from companies, such as Macromolecular Resources (Fort Collins, CO) and Synthegen (Houston, TX).

Also provided herein are isolated or purified nucleic acids comprising a nucleotide sequence which is complementary to the nucleotide sequence of any of the nucleic acids described herein or a nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence of any of the nucleic acids described herein.

The nucleotide sequence which hybridizes under stringent conditions may hybridize under high stringency conditions. The term “high stringency conditions” refers to a nucleotide sequence that specifically hybridizes to a target sequence (the nucleotide sequence of any of the nucleic acids described herein) in an amount that is detectably stronger than non-specific hybridization. High stringency conditions include conditions which would distinguish a polynucleotide with an exact complementary sequence, or one containing only a few scattered mismatches from a random sequence that happened to have a few small regions (e.g., 3-10 bases) that matched the nucleotide sequence. Such small regions of complementarity are more easily melted than a full-length complement of 14-17 or more bases, and high stringency hybridization makes them easily distinguishable. Relatively high stringency conditions would include, for example, low salt and/or high temperature conditions, such as provided by about 0.02-0.1 M NaCl or the equivalent, at temperatures of about 50-70 °C. Such high stringency conditions tolerate little, if any, mismatch between the nucleotide sequence and the template or target strand, and are particularly suitable for detecting expression of any of the CARs described herein. It is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide.

The present disclosure also provides nucleic acids comprising a nucleotide sequence that is at least about 70% or more, e.g., about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to any of the nucleic acids described herein, such as any one of SEQ ID NOs: 32-39.

Nucleic acid sequences of exemplary fusion polypeptides of the present disclosure are provided below.

Exemplary nucleic acid sequence of Construct A: FokI (D450) - polypeptide linker - Fokll

(D450A) - XTEN linker - AsCpfl (A) (SEQ ID NO: 32) atgcaactggtgaagagcgagctggaagagaagaaaagcgagctcagacataagctgaag tacgttccccacgaa tacattgaactgatagaaatcgctagaaacagtacgcaagacagaatactggaaatgaag gtgatggagttcttc atgaaggtttacggctatcgtggcaaacacctcgggggctcccggaagcccgacggggct atctacaccgtgggc agtcccatcgactatggcgtgatcgtggacaccaaagcttatagcggcggatataatctc cccatcggccaagcc gatgagatgcagaggtatgtggaggagaaccaaacaagaaacaagcatatcaaccccaac gagtggtggaaggtt tatcctagctcggtgaccgagtttaagttcctattcgtgtctggccacttcaagggcaac tataaggcacagctc actagactgaatcatatcacgaattgcaacggcgccgtgttatccgtggaggagctactg atcggcggagagatg atcaaagccggcaccctgaccctggaagaggtgagaagaaagtttaacaatggcgaaata aatttcggcagcgga agtggaagcggctccatcactagaaccaccaaccctagaaacgtggtgcccaagatctac atgagcgccggcagc atccccctgaccacccacatcaccaactcaattcagcccaccctgtggaccatcggcagc atcaacggcgtggcc cccctggccaagagcatcaagctgggcatccccgtgaccggcagcgcctacaccgatcag accaccgccatggtg agaaagaaggtgagcgtgttcatgggcagcggcagcgggagcggctcatcgcagctggtt aagagcgagttagaa gaaaaaaagagcgaactgcggcataaactgaagtatgtcccacacgagtacatcgaactg atcgagatcgcgaga aactctacccaagacagaattctggagatgaaagtaatggaatttttcatgaaggtgtat ggatatagagggaag cacctgggtggcagcagaaaacccgccggcgccatctacactgtggggagccccatagac tatggtgtgatcgtg gataccaaggcgtatagcggcggttacaatctgcccattgggcaagcggacgagatgcaa agatatgtggaagag aatcagacgaggaacaagcacattaaccctaatgagtggtggaaggtctaccctagctcc gttaccgagttcaag ttcctgtttgtgagcgggcattttaagggcaactacaaggcacagctgacccgcctgaac cacataacaaactgc aacggtgccgtgctgagcgtagaagagttgctaatcggcggcgagatgatcaaggccggc acgctaaccctcgaa gaggtgcgcagaaagttcaataacggcgaaatcaatttcagcggcagcgagactcccggg acctcagagtccgcc acacccgaaagtacacagttcgagggctttaccaacctgtatcaggtgagcaagacactg cggtttgagctgatc ccacagggcaagaccctgaagcacatccaggagcagggcttcatcgaggaggacaaggcc cgcaatgatcactac aaggagctgaagcccatcatcgatcggatctacaagacctatgccgaccagtgcctgcag ctggtgcagctggat tgggagaacctgagcgccgccatcgactcct at agaaaggagaaaaccgaggagacaaggaacgccct gategag gagcaggccacatatcgcaatgccatccacgactacttcatcggccggacagacaacctg accgatgccatcaat aagagacacgccgagatctacaagggcctgttcaaggccgagctgtttaatggcaaggtg ctgaagcagctgggc accgtgaccacaaccgagcacgagaacgccctgctgcggagcttcgacaagtttacaacc tacttctccggcttt tatagaaacaggaagaacgtgttcagcgccgaggatatcagcacagccatcccacaccgc atcgtgcaggacaac ttccccaagtttaaggagaattgtcacatcttcacacgcctgatcaccgccgtgcccagc ctgcgggagcacttt gagaacgtgaagaaggccatcggcatcttcgtgagcacctccatcgaggaggtgttttcc ttccctttttataac cagctgctgacacagacccagatcgacctgtataaccagctgctgggaggaatctctcgg gaggcaggcaccgag aagatcaagggcctgaacgaggtgctgaatctggccatccagaagaatgatgagacagcc cacatcatcgcctcc ctgccacacagattcatccccctgtttaagcagatcctgtccgataggaacaccctgtct ttcatcctggaggag tttaagagcgacgaggaagtgatccagtccttctgcaagtacaagacactgctgagaaac gagaacgtgctggag acagccgaggccctgtttaacgagctgaacagcatcgacctgacacacatcttcatcagc cacaagaagctggag acaatcagcagcgccctgtgcgaccactgggatacactgaggaatgccctgtatgagcgg agaatctccgagctg acaggcaagatcaccaagtctgccaaggagaaggtgcagcgcagcctgaagcacgaggat atcaacctgcaggag atcatctctgccgcaggcaaggagctgagcgaggccttcaagcagaaaaccagcgagatc ctgtcccacgcacac gccgccctggatcagccactgcctacaaccctgaagaagcaggaggagaaggagatcctg aagtctcagctggac agcctgctgggcctgtaccacctgctggactggtttgccgtggatgagtccaacgaggtg gaccccgagttctct gcccggctgaccggcatcaagctggagatggagccttctctgagcttctacaacaaggcc agaaattatgccacc aagaagccctactccgtggagaagttcaagctgaactttcagatgcctacactggccaga ggctgggacgtgaat gtggagaagaacagaggcgccatcctgtttgtgaagaacggcctgtactatctgggcatc atgccaaagcagaag ggcaggtataaggccctgagcttcgagcccacagagaaaaccagcgagggctttgataag atgtactatgactac ttccctgatgccgccaagatgatcccaaagtgcagcacccagctgaaggccgtgacagcc cactttcagacccac acaacccccatcctgctgtccaacaatttcatcgagcctctggagatcacaaaggagatc tacgacctgaacaat cctgagaaggagccaaagaagtttcagacagcctacgccaagaaaaccggcgaccagaag ggctacagagaggcc ctgtgcaagtggatcgacttcacaagggattttctgtccaagtataccaagacaacctct atcgatctgtctagc ctgcggccatcctctcagtataaggacctgggcgagtactatgccgagctgaatcccctg ctgtaccacatcagc ttccagagaatcgccgagaaggagatcatggatgccgtggagacaggcaagctgtacctg ttccagatctataac aaggactttgccaagggccaccacggcaagcctaatctgcacacactgtattggaccggc ctgttttctccagag aacctggccaagacaagcatcaagctgaatggccaggccgagctgttctaccgccctaag tccaggatgaagagg atggcacaccggctgggagagaagatgctgaacaagaagctgaaggatcagaaaacccca atccccgacaccctg taccaggagctgtacgactatgtgaatcacagactgtcccacgacctgtctgatgaggcc agggccctgctgccc aacgtgatcaccaaggaggtgtctcacgagatcatcaaggataggcgctttaccagcgac aagttctttttccac gtgcctatcacactgaactatcaggccgccaattccccatctaagttcaaccagagggtg aatgcctacctgaag gagcaccccgagacacctatcatcggcatcgcccggggcgagagaaacctgatctatatc acagtgatcgactcc accggcaagatcctggagcagcggagcctgaacaccatccagcagtttgattaccagaag aagctggacaacagg gagaaggagagggtggcagcaaggcaggcctggtctgtggtgggcacaatcaaggatctg aagcagggctatctg agccaggtcatccacgagatcgtggacctgatgatccactaccaggccgtggtggtgctg gagaacctgaatttc ggctttaagagcaagaggaccggcatcgccgagaaggccgtgtaccagcagttcgagaag atgctgatcgataag ctgaattgcctggtgctgaaggactatccagcagagaaagtgggaggcgtgctgaaccca taccagctgacagac cagttcacctcctttgccaagatgggcacccagtctggcttcctgttttacgtgcctgcc ccatatacatctaag atcgatcccctgaccggcttcgtggaccccttcgtgtggaaaaccatcaagaatcacgag agccgcaagcacttc ctggagggcttcgactttctgcactacgacgtgaaaaccggcgacttcatcctgcacttt aagatgaacagaaat ctgtccttccagaggggcctgcccggctttatgcctgcatgggatatcgtgttcgagaag aacgagacacagttt gacgccaagggcacccctttcatcgccggcaagagaatcgtgccagtgatcgagaatcac agattcaccggcaga taccgggacctgtatcctgccaacgagctgatcgccctgctggaggagaagggcatcgtg ttcagggatggctcc aacatcctgccaaagctgctggagaatgacgattctcacgccatcgacaccatggtggcc ctgatccgcagcgtg ctgcagatgcggaactccaatgccgccacaggcgaggactatatcaacagccccgtgcgc gatctgaatggcgtg tgcttcgactcccggtttcagaacccagagtggcccatggacgccgatgccaatggcgcc taccacatcgccctg aagggccagctgctgctgaatcacctgaaggagagcaaggatctgaagctgcagaacggc atctccaatcaggac tggctggcctacatccaggagctgcgcaac

Exemplary nucleic acid sequence of Construct B: APOBEC Base Editor - AsCpfl (D908A)

- XTEN - FokI (D450A) - polypeptide linker - Fokll (D450) (SEQ ID NO: 33) atgggcagctcagagactggcccagtggctgtggaccccacattgagacggcggatcgag ccccatgagtttgag gtattcttcgatccgagagagctccgcaaggagacctgcctgctttacgaaattaattgg gggggccggcactcc atttggcgacatacatcacagaacactaacaagcacgtcgaagtcaacttcatcgagaag ttcacgacagaaaga tatttctgtccgaacacaaggtgcagcattacctggtttctcagctggagcccatgcggc gaatgtagtagggcc atcactgaattcctgtcaaggtatccccacgtcactctgtttatttacatcgcaaggctg taccaccacgctgac ccccgcaatcgacaaggcctgcgggatttgatctcttcaggtgtgactatccaaattatg actgagcaggagtca ggatactgctggagaaactttgtgaattatagcccgagtaatgaagcccactggcctagg tatccccatctgtgg gtacgactgtacgttcttgaactgtactgcatcatactgggcctgcctccttgtctcaac attctgagaaggaag cagccacagctgacattctttaccatcgctcttcagtcttgtcattaccagcgactgccc ccacacattctctgg gccaccgggttgaaatctggtggttcttctggtggttctagcggcagcgagactcccggg acctcagagtccgcc acacccgaaagttccggagggagtagcggcgggtctacacagttcgagggctttaccaac ctgtatcaggtgagc aagacactgcggtttgagctgatcccacagggcaagaccctgaagcacatccaggagcag ggcttcatcgaggag gacaaggcccgcaatgatcactacaaggagctgaagcccatcatcgatcggatctacaag acctatgccgaccag tgcctgcagctggtgcagctggattgggagaacctgagcgccgccatcgactcctataga aaggagaaaaccgag gagacaaggaacgccctgatcgaggagcaggccacatatcgcaatgccatccacgactac ttcatcggccggaca gacaacctgaccgatgccatcaataagagacacgccgagatctacaagggcctgttcaag gccgagctgtttaat ggcaaggtgctgaagcagctgggcaccgtgaccacaaccgagcacgagaacgccctgctg cggagcttcgacaag tttacaacctacttctccggcttttatagaaacaggaagaacgtgttcagcgccgaggat atcagcacagccatc ccacaccgcatcgtgcaggacaacttccccaagtttaaggagaattgtcacatcttcaca cgcctgatcaccgcc gtgcccagcctgcgggagcactttgagaacgtgaagaaggccatcggcatcttcgtgagc acctccatcgaggag gtgttttccttccctttttataaccagctgctgacacagacccagatcgacctgtataac cagctgctgggagga atctctcgggaggcaggcaccgagaagatcaagggcctgaacgaggtgctgaatctggcc atccagaagaatgat gagacagcccacatcatcgcctccctgccacacagattcatccccctgtttaagcagatc ctgtccgataggaac accctgtctttcatcctggaggagtttaagagcgacgaggaagtgatccagtccttctgc aagtacaagacactg ctgagaaacgagaacgtgctggagacagccgaggccctgtttaacgagctgaacagcatc gacctgacacacatc ttcatcagccacaagaagctggagacaatcagcagcgccctgtgcgaccactgggataca ctgaggaatgccctg tatgagcggagaatctccgagctgacaggcaagatcaccaagtctgccaaggagaaggtg cagcgcagcctgaag cacgaggatatcaacctgcaggagatcatctctgccgcaggcaaggagctgagcgaggcc ttcaagcagaaaacc agcgagatcctgtcccacgcacacgccgccctggatcagccactgcctacaaccctgaag aagcaggaggagaag gagatcctgaagtctcagctggacagcctgctgggcctgtaccacctgctggactggttt gccgtggatgagtcc aacgaggtggaccccgagttctctgcccggctgaccggcatcaagctggagatggagcct tctctgagcttctac aacaaggccagaaattatgccaccaagaagccctactccgtggagaagttcaagctgaac tttcagatgcctaca ctggccagaggctgggacgtgaatgtggagaagaacagaggcgccatcctgtttgtgaag aacggcctgtactat ctgggcatcatgccaaagcagaagggcaggtataaggccctgagcttcgagcccacagag aaaaccagcgagggc tttgataagatgtactatgactacttccctgatgccgccaagatgatcccaaagtgcagc acccagctgaaggcc gtgacagcccactttcagacccacacaacccccatcctgctgtccaacaatttcatcgag cctctggagatcaca aaggagatctacgacctgaacaatcctgagaaggagccaaagaagtttcagacagcctac gccaagaaaaccggc gaccagaagggctacagagaggccctgtgcaagtggatcgacttcacaagggattttctg tccaagtataccaag acaacctctatcgatctgtctagcctgcggccatcctctcagtataaggacctgggcgag tactatgccgagctg aatcccctgctgtaccacatcagcttccagagaatcgccgagaaggagatcatggatgcc gtggagacaggcaag ctgtacctgttccagatctataacaaggactttgccaagggccaccacggcaagcctaat ctgcacacactgtat tggaccggcctgttttctccagagaacctggccaagacaagcatcaagctgaatggccag gccgagctgttctac cgccctaagtccaggatgaagaggatggcacaccggctgggagagaagatgctgaacaag aagctgaaggatcag aaaaccccaatccccgacaccctgtaccaggagctgtacgactatgtgaatcacagactg tcccacgacctgtct gatgaggccagggccctgctgcccaacgtgatcaccaaggaggtgtctcacgagatcatc aaggataggcgcttt accagcgacaagttctttttccacgtgcctatcacactgaactatcaggccgccaattcc ccatctaagttcaac cagagggtgaatgcctacctgaaggagcaccccgagacacctatcatcggcatcgcccgg ggcgagagaaacctg atctatatcacagtgatcgactccaccggcaagatcctggagcagcggagcctgaacacc atccagcagtttgat taccagaagaagctggacaacagggagaaggagagggtggcagcaaggcaggcctggtct gtggtgggcacaatc aaggatctgaagcagggctatctgagccaggtcatccacgagatcgtggacctgatgatc cactaccaggccgtg gtggtgctggagaacctgaatttcggctttaagagcaagaggaccggcatcgccgagaag gccgtgtaccagcag ttcgagaagatgctgatcgataagctgaattgcctggtgctgaaggactatccagcagag aaagtgggaggcgtg ctgaacccataccagctgacagaccagttcacctcctttgccaagatgggcacccagtct ggcttcctgttttac gtgcctgccccatatacatctaagatcgatcccctgaccggcttcgtggaccccttcgtg tggaaaaccatcaag aatcacgagagccgcaagcacttcctggagggcttcgactttctgcactacgacgtgaaa accggcgacttcatc ctgcactttaagatgaacagaaatctgtccttccagaggggcctgcccggctttatgcct gcatgggatatcgtg ttcgagaagaacgagacacagtttgacgccaagggcaccccttt cat cgccggcaagagaatcgtgccagt gate gagaatcacagattcaccggcagataccgggacctgtatcctgccaacgagctgatcgcc ctgctggaggagaag ggcatcgtgttcagggatggctccaacatcctgccaaagctgctggagaatgacgattct cacgccatcgacacc atggtggccctgatccgcagcgtgctgcagatgcggaactccaatgccgccacaggcgag gactatatcaacagc cccgtgcgcgatctgaatggcgtgtgcttcgactcccggtttcagaacccagagtggccc atggacgccgatgcc aatggcgcctaccacatcgccctgaagggccagctgctgctgaatcacctgaaggagagc aaggatctgaagctg cagaacggcatctccaatcaggactggctggcctacatccaggagctgcgcaacagcggc agcgagactcccggg acctcagagtccgccacacccgaaagtcaactggtgaagagcgagctggaagagaagaaa agcgagctcagacat aagctgaagtacgttccccacgaatacattgaact gat agaaat eget agaaacagtacgcaagacagaat act g gaaatgaaggtgatggagttcttcatgaaggtttacggctatcgtggcaaacacctcggg ggctcccggaagccc gccggggctatctacaccgtgggcagtcccatcgactatggcgtgatcgtggacaccaaa gcttatagcggcgga tataatctccccatcggccaagccgatgagatgcagaggtatgtggaggagaaccaaaca agaaacaagcatatc aaccccaacgagtggtggaaggtttatcctagctcggtgaccgagtttaagttcctattc gtgtctggccacttc aagggcaactataaggcacagctcactagactgaatcatatcacgaattgcaacggcgcc gtgttatccgtggag gagetact gat eggeggagagat gat caaagccggcaccctgaccctggaagaggtgagaagaaagtttaacaat ggcgaaataaatttcggcagcggaagtggaagcggctccatcactagaaccaccaaccct agaaacgtggtgccc aagatctacatgagcgccggcagcatccccctgaccacccacatcaccaactcaattcag cccaccctgtggacc atcggcagcatcaacggcgtggcccccctggccaagagcatcaagctgggcatccccgtg accggcagcgcctac accgatcagaccaccgccatggtgagaaagaaggtgagcgtgttcatgggcagcggcagc gggagcggctcatcg cagctggttaagagcgagttagaagaaaaaaagagcgaactgcggcataaactgaagtat gtcccacacgagtac atcgaactgatcgagatcgcgagaaactctacccaagacagaattctggagatgaaagta atggaatttttcatg aaggtgtatggatatagagggaagcacctgggtggcagcagaaaacccgacggcgccatc tacactgtggggagc cccatagactatggtgtgatcgtggataccaaggcgtatagcggcggttacaatctgccc attgggcaagcggac gagatgcaaagatatgtggaagagaatcagacgaggaacaagcacattaaccctaatgag tggtggaaggtctac cctagctccgttaccgagttcaagttcctgtttgtgagcgggcattttaagggcaactac aaggcacagctgacc cgcctgaaccacataacaaactgcaacggtgccgtgctgagcgtagaagagttgctaatc ggcggcgagatgatc aaggccggcacgctaaccctcgaagaggtgcgcagaaagttcaataacggcgaaatcaat ttc

Exemplary nucleic acid sequence of Construct C: APOBEC Base Editor - MAD7™ (D908A)

- XTEN - FokI (D450A) - polypeptide linker - Fokll (D450) (SEQ ID NO: 34) atgggcagctcagagactggcccagtggctgtggaccccacattgagacggcggatcgag ccccatgagtttgag gtattcttcgatccgagagagctccgcaaggagacctgcctgctttacgaaattaattgg gggggccggcactcc atttggcgacatacatcacagaacactaacaagcacgtcgaagtcaacttcatcgagaag ttcacgacagaaaga tatttctgtccgaacacaaggtgcagcattacctggtttctcagctggagcccatgcggc gaatgtagtagggcc atcactgaattcctgtcaaggtatccccacgtcactctgtttatttacatcgcaaggctg taccaccacgctgac ccccgcaatcgacaaggcctgcgggatttgatctcttcaggtgtgactatccaaattatg actgagcaggagtca ggatactgctggagaaactttgtgaattatagcccgagtaatgaagcccactggcctagg tatccccatctgtgg gtacgactgtacgttcttgaactgtactgcatcatactgggcctgcctccttgtctcaac attctgagaaggaag cagccacagctgacattctttaccatcgctcttcagtcttgtcattaccagcgactgccc ccacacattctctgg gccaccgggttgaaatctggtggttcttctggtggttctagcggcagcgagactcccggg acctcagagtccgcc acacccgaaagttccggagggagtagcggcgggtctaataacggaactaataacttccaa aacttcatcgggatc agttccttgcagaaaactctccggaatgctctcatcccaactgagactactcagcagttc attgttaagaatgga atcataaaagaggacgagcttaggggggaaaataggcaaatcctcaaggatatcatggat gactattataggggc tttatatccgagacactgagcagcattgatgatatagactggacctctcttttcgaaaag atggaaatacaactt aaaaatggagataacaaggacaccctgataaaggaacagaccgaatataggaaggcaatt cataaaaagtttgct aacgatgataggtttaaaaacatgttctcagcaaaactcatttcagatatactgcccgaa ttcgttatccacaac aacaactactccgctagcgaaaaagaggaaaagacccaagtcataaagctgttctctcga ttcgcgacgagtttt aaagattatttccgaaatcgcgcaaactgtttctcagctgatgatatcagcagctcatcc tgtcatcggatcgtt aacgataatgctgaaatcttcttctccaatgcacttgtttataggcgcattgttaaatct ctctcaaacgatgat atcaataagatttccggcgatatgaaggacagtcttaaggagatgagcctcgaagagata tactcatacgagaaa tatggcgaatttatcacccaggaagggatttccttctataatgacatttgcggcaaagtc aattccttcatgaac ctgtattgccaaaaaaataaagaaaacaagaacctctataagctgcaaaagttgcataag caaatactttgtatc gcggatacaagctatgaagttccctacaagttcgagagtgatgaggaggtgtatcaatct gtcaatggtttcctt gataatatttcttctaagcatattgttgaacgactccgaaagataggagacaactataat ggatacaatttggat aaaatctacatcgtgtctaaattttacgagagtgtgtcacaaaaaacatatagagactgg gagacaattaatacc gccctggagatacattacaacaatatacttcccgggaacgggaagtctaaggcagacaag gtgaagaaagccgtg aagaacgacttgcaaaagtcaattaccgaaatcaatgagcttgtttcaaactataaactt tgttcagatgacaat attaaagccgaaacctatattcatgaaatctctcatattctgaataactttgaggcgcaa gaactgaaatataac ccagaaatacacctcgttgagtccgaactgaaagcaagcgaactgaaaaatgttttggac gtgataatgaacgct tttcattggtgctcagtctttatgacagaggagcttgttgacaaggataacaatttctat gcggaactggaagag atttacgacgaaatctatccggtcatatccctgtataacctggttcgcaactatgtcacg caaaaaccatacagc acgaagaagattaaactgaactttggtattccgacgctggcccgcggatggtcaaaatct gttgaatactcacga aatgccataatcctgatgcgagataacctctactaccttggaatctttaatgctaaaaat aaacccgataaaaaa attatcgaagggaacacgagtgaaaacaaaggtgattataaaaaaatgatatataatctg cttccaggaccaaat aagatgatacccaaagttttcctttcttcaaagaccggcgtcgagacatataaaccatcc gcgtacatacttgaa ggctacaaacaaaataaacatatcaaatcatctaaggattttgacattacgttctgtcat gatttgattgactat ttcaaaaattgcatagccattcatccagagtggaaaaactttgggtttgacttctctgat accagtacatatgaa gacataagtggattttaccgagaagtagagctccaaggttataaaatagactggacctat atatctgaaaaggat atagaccttttgcaagagaagggacagctttatcttttccaaatctacaacaaagacttc agtaagaaaagtacc gggaatgacaatcttcataccatgtatctgaagaacctgttctccgaagaaaatctgaag gacatagtcctgaag cttaatggcgaagcggaaatttttttccgaaagagctctattaagaaccccataatacat aagaagggaagcatt ctcgttaatcgaacgtatgaggccgaagagaaagatcaatttgggaatatccaaatcgtt cgaaagaacatacca gaaaatatttaccaagaattgtacaaatattttaacgataaaagcgacaaagaactgtct gatgaagctgctaag ctgaaaaacgtcgtcggccatcatgaggccgcgacgaatatagtcaaggattaccgatat acatacgataagtat ttcctgcatatgcccatcactatcaactttaaggcaaataagactggattcattaatgac agaatactgcaatac atagctaaagaaaaagatttgcatgttattggcattgccaggggtgagcgcaatcttatc tatgtaagcgtcatt gatacttgcgggaatatcgtagagcagaagtcatttaatattgtaaatgggtacgattac caaatcaagttgaag cagcaagagggagcacgacagattgcccgcaaggagtggaaagagatcggaaagataaag gagatcaaggagggg tatttgtcccttgttatacacgaaatttccaagatggtaatcaagtacaacgctataatt gctatggaggatctc tcctatggatttaaaaagggaagatttaaagtcgagcggcaggtatatcagaaatttgaa acaatgcttattaat aaacttaattatctcgttttcaaagacattagtatcaccgaaaacggtgggctgttgaag ggctatcaacttacg tacataccagataagcttaagaatgtgggtcaccaatgcggatgcatattctacgtgccc gcagcttatacaagc aaaatcgacccaacaacgggtttcgtaaacatatttaagttcaaggatctcaccgtggat gccaagcgagagttc ataaaaaaatttgactcaatcagatatgactcagaaaagaatcttttttgttttaccttc gactacaataatttc attacacaaaatacggttatgagcaagtcatcctggtccgtatatacgtatggagtgcgc ataaagcggagattc gttaacgggcgattttctaatgagtccgatacaatcgatataacaaaggatatggaaaaa actctggaaatgact gatataaattggagggacggtcatgacctcaggcaagacattatcgattatgagatcgtg caacatatttttgag atctttcggttgactgtccaaatgaggaactctctgtctgaattggaagatagggactac gatcgcctgataagc cccgtgttgaacgagaataacatattctacgattccgcgaaagccggggatgcgctccct aaggacgccgatgca aatggggcctattgtattgctttgaaagggctgtacgaaatcaaacagatcaccgaaaac tggaaagaagacggg aagtttagtcgggataaactgaagatatccaacaaggactggtttgactttatccaaaat aagcgatatttgagc ggcagcgagactcccgggacctcagagtccgccacacccgaaagtcaactggtgaagagc gagctggaagagaag aaaagcgagctcagacataagctgaagtacgttccccacgaatacattgaactgatagaa atcgctagaaacagt acgcaagacagaatactggaaatgaaggtgatggagttcttcatgaaggtttacggctat cgtggcaaacacctc gggggctcccggaagcccgccggggctatctacaccgtgggcagtcccatcgactatggc gtgatcgtggacacc aaagcttatagcggcggatataatctccccatcggccaagccgatgagatgcagaggtat gtggaggagaaccaa acaagaaacaagcatatcaaccccaacgagtggtggaaggtttatcctagctcggtgacc gagtttaagttccta ttcgtgtctggccacttcaagggcaactataaggcacagctcactagactgaatcatatc acgaattgcaacggc gccgtgttatccgtggaggagctactgatcggcggagagatgatcaaagccggcaccctg accctggaagaggtg agaagaaagtttaacaatggcgaaataaatttcggcagcggaagtggaagcggctccatc actagaaccaccaac cctagaaacgtggtgcccaagatctacatgagcgccggcagcatccccctgaccacccac atcaccaactcaatt cagcccaccctgtggaccatcggcagcatcaacggcgtggcccccctggccaagagcatc aagctgggcatcccc gtgaccggcagcgcctacaccgatcagaccaccgccatggtgagaaagaaggtgagcgtg ttcatgggcagcggc agcgggagcggctcatcgcagctggttaagagcgagttagaagaaaaaaagagcgaactg cggcataaactgaag tatgtcccacacgagtacatcgaactgatcgagatcgcgagaaactctacccaagacaga attctggagatgaaa gtaatggaatttttcatgaaggtgtatggatatagagggaagcacctgggtggcagcaga aaacccgacggcgcc atctacactgtggggagccccatagactatggtgtgatcgtggataccaaggcgtatagc ggcggttacaatctg cccattgggcaagcggacgagatgcaaagatatgtggaagagaatcagacgaggaacaag cacattaaccctaat gagtggtggaaggtctaccctagctccgttaccgagttcaagttcctgtttgtgagcggg cattttaagggcaac tacaaggcacagctgacccgcctgaaccacataacaaactgcaacggtgccgtgctgagc gtagaagagttgcta atcggcggcgagatgatcaaggccggcacgctaaccctcgaagaggtgcgcagaaagttc aataacggcgaaatc aatttc

Exemplary nucleic acid sequence of Construct E: Cpfl (D908A) -XTEN - FokI- polypeptide linker - Fokll (SEQ ID NO: 35) atgacacagttcgagggctttaccaacctgtatcaggtgagcaagacactgcggtttgag ctgatcccacagggc aagaccctgaagcacatccaggagcagggcttcatcgaggaggacaaggcccgcaatgat cactacaaggagctg aagcccatcatcgatcggatctacaagacctatgccgaccagtgcctgcagctggtgcag ctggattgggagaac ctgagcgccgccatcgactcctatagaaaggagaaaaccgaggagacaaggaacgccctg atcgaggagcaggcc acatatcgcaatgccatccacgactacttcatcggccggacagacaacctgaccgatgcc atcaataagagacac gccgagatctacaagggcctgttcaaggccgagctgtttaatggcaaggtgctgaagcag ctgggcaccgtgacc acaaccgagcacgagaacgccctgctgcggagcttcgacaagtttacaacctacttctcc ggcttttatagaaac aggaagaacgtgttcagcgccgaggatatcagcacagccatcccacaccgcatcgtgcag gacaacttccccaag tttaaggagaattgtcacatcttcacacgcctgatcaccgccgtgcccagcctgcgggag cactttgagaacgtg aagaaggccatcggcatcttcgtgagcacctccatcgaggaggtgttttccttccctttt tataaccagctgctg acacagacccagatcgacctgtataaccagctgctgggaggaatctctcgggaggcaggc accgagaagatcaag ggcctgaacgaggtgctgaatctggccatccagaagaatgatgagacagcccacatcatc gcctccctgccacac agattcatccccctgtttaagcagatcctgtccgataggaacaccctgtctttcatcctg gaggagtttaagagc gacgaggaagtgatccagtccttctgcaagtacaagacactgctgagaaacgagaacgtg ctggagacagccgag gccctgtttaacgagctgaacagcatcgacctgacacacatcttcatcagccacaagaag ctggagacaatcagc agcgccctgtgcgaccactgggatacactgaggaatgccctgtatgagcggagaatctcc gagctgacaggcaag atcaccaagtctgccaaggagaaggtgcagcgcagcctgaagcacgaggatatcaacctg caggagatcatctct gccgcaggcaaggagctgagcgaggccttcaagcagaaaaccagcgagatcctgtcccac gcacacgccgccctg gatcagccactgcctacaaccctgaagaagcaggaggagaaggagatcctgaagtctcag ctggacagcctgctg ggcctgtaccacctgctggactggtttgccgtggatgagtccaacgaggtggaccccgag ttctctgcccggctg accggcatcaagctggagatggagccttctctgagcttctacaacaaggccagaaattat gccaccaagaagccc tactccgtggagaagttcaagctgaactttcagatgcctacactggccagaggctgggac gtgaatgtggagaag aacagaggcgccatcctgtttgtgaagaacggcctgtactatctgggcatcatgccaaag cagaagggcaggtat aaggccctgagcttcgagcccacagagaaaaccagcgagggctttgataagatgtactat gactacttccctgat gccgccaagatgatcccaaagtgcagcacccagctgaaggccgtgacagcccactttcag acccacacaaccccc atcctgctgtccaacaatttcatcgagcctctggagatcacaaaggagatctacgacctg aacaatcctgagaag gagccaaagaagtttcagacagcctacgccaagaaaaccggcgaccagaagggctacaga gaggccctgtgcaag tggatcgacttcacaagggattttctgtccaagtataccaagacaacctctatcgatctg tctagcctgcggcca tcctctcagtataaggacctgggcgagtactatgccgagctgaatcccctgctgtaccac atcagcttccagaga atcgccgagaaggagatcatggatgccgtggagacaggcaagctgtacctgttccagatc tataacaaggacttt gccaagggccaccacggcaagcctaatctgcacacactgtattggaccggcctgttttct ccagagaacctggcc aagacaagcatcaagctgaatggccaggccgagctgttctaccgccctaagtccaggatg aagaggatggcacac cggctgggagagaagatgctgaacaagaagctgaaggatcagaaaaccccaatccccgac accctgtaccaggag ctgtacgactatgtgaatcacagactgtcccacgacctgtctgatgaggccagggccctg ctgcccaacgtgatc accaaggaggtgtctcacgagatcatcaaggataggcgctttaccagcgacaagttcttt ttccacgtgcctatc acactgaactatcaggccgccaattccccatctaagttcaaccagagggtgaatgcctac ctgaaggagcacccc gagacacctatcatcggcatcgcccggggcgagagaaacctgatctatatcacagtgatc gactccaccggcaag atcctggagcagcggagcctgaacaccatccagcagtttgattaccagaagaagctggac aacagggagaaggag agggtggcagcaaggcaggcctggtctgtggtgggcacaatcaaggatctgaagcagggc tatctgagccaggtc atccacgagatcgtggacctgatgatccactaccaggccgtggtggtgctggagaacctg aatttcggctttaag agcaagaggaccggcatcgccgagaaggccgtgtaccagcagttcgagaagatgctgatc gataagctgaattgc ctggtgctgaaggactatccagcagagaaagtgggaggcgtgctgaacccataccagctg acagaccagttcacc tcctttgccaagatgggcacccagtctggcttcctgttttacgtgcctgccccatataca tctaagatcgatccc ctgaccggcttcgtggaccccttcgtgtggaaaaccatcaagaatcacgagagccgcaag cacttcctggagggc ttcgactttctgcactacgacgtgaaaaccggcgacttcatcctgcactttaagatgaac agaaatctgtccttc cagaggggcctgcccggctttatgcctgcatgggatatcgtgttcgagaagaacgagaca cagtttgacgccaag ggcacccctttcatcgccggcaagagaatcgtgccagtgatcgagaatcacagattcacc ggcagataccgggac ctgtatcctgccaacgagctgatcgccctgctggaggagaagggcatcgtgttcagggat ggctccaacatcctg ccaaagctgctggagaatgacgattctcacgccatcgacaccatggtggccctgatccgc agcgtgctgcagatg cggaactccaatgccgccacaggcgaggactatatcaacagccccgtgcgcgatctgaat ggcgtgtgcttcgac tcccggtttcagaacccagagtggcccatggacgccgatgccaatggcgcctaccacatc gccctgaagggccag ctgctgctgaatcacctgaaggagagcaaggatctgaagctgcagaacggcatctccaat caggactggctggcc tacatccaggagctgcgcaacagcggcagcgagactcccgggacctcagagtccgccaca cccgaaagtcaactg gtgaagagcgagctggaagagaagaaaagcgagctcagacataagctgaagtacgttccc cacgaatacattgaa ctgatagaaatcgctagaaacagtacgcaagacagaatactggaaatgaaggtgatggag ttcttcatgaaggtt tacggctatcgtggcaaacacctcgggggctcccggaagcccgacggggctatctacacc gtgggcagtcccatc gactatggcgtgatcgtggacaccaaagcttatagcggcggatataatctccccatcggc caagccgatgagatg cagaggtatgtggaggagaaccaaacaagaaacaagcatatcaaccccaacgagtggtgg aaggtttatcctagc tcggtgaccgagtttaagttcctattcgtgtctggccacttcaagggcaactataaggca cagctcactagactg aatcatatcacgaattgcaacggcgccgtgttatccgtggaggagctactgatcggcgga gagatgatcaaagcc ggcaccctgaccctggaagaggtgagaagaaagtttaacaatggcgaaataaatttcggc agcggaagtggaagc ggctccatcactagaaccaccaaccctagaaacgtggtgcccaagatctacatgagcgcc ggcagcatccccctg accacccacatcaccaactcaattcagcccaccctgtggaccatcggcagcatcaacggc gtggcccccctggcc aagagcatcaagctgggcatccccgtgaccggcagcgcctacaccgatcagaccaccgcc atggtgagaaagaag gtgagcgtgttcatgggcagcggcagcgggagcggctcatcgcagctggttaagagcgag ttagaagaaaaaaag agcgaactgcggcataaactgaagtatgtcccacacgagtacatcgaactgatcgagatc gcgagaaactctacc caagacagaattctggagatgaaagtaatggaatttttcatgaaggtgtatggatataga gggaagcacctgggt ggcagcagaaaacccgacggcgccatctacactgtggggagccccatagactatggtgtg atcgtggataccaag gcgtatagcggcggttacaatctgcccattgggcaagcggacgagatgcaaagatatgtg gaagagaatcagacg aggaacaagcacattaaccctaatgagtggtggaaggtctaccctagctccgttaccgag ttcaagttcctgttt gtgagcgggcattttaagggcaactacaaggcacagctgacccgcctgaaccacataaca aactgcaacggtgcc gtgctgagcgtagaagagttgctaatcggcggcgagatgatcaaggccggcacgctaacc ctcgaagaggtgcgc agaaagttcaataacggcgaaatcaatttc

Exemplary nucleic acid sequence of Construct F: FokI- polypeptide linker - Fokll - XTEN -

Cpfl (D908A) (SEQ ID NO: 36) atgcaactggtgaagagcgagctggaagagaagaaaagcgagctcagacataagctgaag tacgttccccacgaa tacattgaactgatagaaatcgctagaaacagtacgcaagacagaatactggaaatgaag gtgatggagttcttc atgaaggtttacggctatcgtggcaaacacctcgggggctcccggaagcccgacggggct atctacaccgtgggc agtcccatcgactatggcgtgatcgtggacaccaaagcttatagcggcggatataatctc cccatcggccaagcc gatgagatgcagaggtatgtggaggagaaccaaacaagaaacaagcatatcaaccccaac gagtggtggaaggtt tatcctagctcggtgaccgagtttaagttcctattcgtgtctggccacttcaagggcaac tataaggcacagctc actagactgaatcatatcacgaattgcaacggcgccgtgttatccgtggaggagctactg atcggcggagagatg atcaaagccggcaccctgaccctggaagaggtgagaagaaagtttaacaatggcgaaata aatttcggcagcgga agtggaagcggctccatcactagaaccaccaaccctagaaacgtggtgcccaagatctac atgagcgccggcagc atccccctgaccacccacatcaccaactcaattcagcccaccctgtggaccatcggcagc atcaacggcgtggcc cccctggccaagagcatcaagctgggcatccccgtgaccggcagcgcctacaccgatcag accaccgccatggtg agaaagaaggtgagcgtgttcatgggcagcggcagcgggagcggctcatcgcagctggtt aagagcgagttagaa gaaaaaaagagcgaactgcggcataaactgaagtatgtcccacacgagtacatcgaactg atcgagatcgcgaga aactctacccaagacagaattctggagatgaaagtaatggaatttttcatgaaggtgtat ggatatagagggaag cacctgggtggcagcagaaaacccgacggcgccatctacactgtggggagccccatagac tatggtgtgatcgtg gataccaaggcgtatagcggcggttacaatctgcccattgggcaagcggacgagatgcaa agatatgtggaagag aatcagacgaggaacaagcacattaaccctaatgagtggtggaaggtctaccctagctcc gttaccgagttcaag ttcctgtttgtgagcgggcattttaagggcaactacaaggcacagctgacccgcctgaac cacataacaaactgc aacggtgccgtgctgagcgtagaagagttgctaatcggcggcgagatgatcaaggccggc acgctaaccctcgaa gaggtgcgcagaaagttcaataacggcgaaatcaatttcagcggcagcgagactcccggg acctcagagtccgcc acacccgaaagtacacagttcgagggctttaccaacctgtatcaggtgagcaagacactg cggtttgagctgatc ccacagggcaagaccctgaagcacatccaggagcagggcttcatcgaggaggacaaggcc cgcaatgatcactac aaggagctgaagcccatcatcgatcggatctacaagacctatgccgaccagtgcctgcag ctggtgcagctggat tgggagaacctgagcgccgccatcgactcct at agaaaggagaaaaccgaggagacaaggaacgccct gategag gagcaggccacatatcgcaatgccatccacgactacttcatcggccggacagacaacctg accgatgccatcaat aagagacacgccgagatctacaagggcctgttcaaggccgagctgtttaatggcaaggtg ctgaagcagctgggc accgtgaccacaaccgagcacgagaacgccctgctgcggagcttcgacaagtttacaacc tacttctccggcttt tatagaaacaggaagaacgtgttcagcgccgaggatatcagcacagccatcccacaccgc atcgtgcaggacaac ttccccaagtttaaggagaattgtcacatcttcacacgcctgatcaccgccgtgcccagc ctgcgggagcacttt gagaacgtgaagaaggccat eggcat cttcgtgagcacctccatcgaggaggtgttttccttcccttttt at aac cagctgctgacacagacccagatcgacctgtataaccagctgctgggaggaatctctcgg gaggcaggcaccgag aagatcaagggcctgaacgaggtgctgaatctggccatccagaagaatgatgagacagcc cacatcatcgcctcc ctgccacacagattcatccccctgtttaagcagatcctgtccgataggaacaccctgtct ttcatcctggaggag tttaagagcgacgaggaagtgatccagtccttctgcaagtacaagacactgctgagaaac gagaacgtgctggag acagccgaggccctgtttaacgagctgaacagcatcgacctgacacacatcttcatcagc cacaagaagctggag acaatcagcagcgccctgtgcgaccactgggatacactgaggaatgccctgtatgagcgg agaatctccgagctg acaggcaagatcaccaagtctgccaaggagaaggtgcagcgcagcctgaagcacgaggat atcaacctgcaggag atcatctctgccgcaggcaaggagctgagcgaggccttcaagcagaaaaccagcgagatc ctgtcccacgcacac gccgccctggatcagccactgcctacaaccctgaagaagcaggaggagaaggagatcctg aagtctcagctggac agcctgctgggcctgtaccacctgctggactggtttgccgtggatgagtccaacgaggtg gaccccgagttctct gcccggctgaccggcatcaagctggagatggagccttctctgagcttctacaacaaggcc agaaattatgccacc aagaagccctactccgtggagaagttcaagctgaactttcagatgcctacactggccaga ggctgggacgtgaat gtggagaagaacagaggcgccatcctgtttgtgaagaacggcctgtactatctgggcatc atgccaaagcagaag ggcaggtataaggccctgagcttcgagcccacagagaaaaccagcgagggctttgataag atgtactatgactac ttccctgatgccgccaagatgatcccaaagtgcagcacccagctgaaggccgtgacagcc cactttcagacccac acaacccccatcctgctgtccaacaatttcatcgagcctctggagatcacaaaggagatc tacgacctgaacaat cctgagaaggagccaaagaagtttcagacagcctacgccaagaaaaccggcgaccagaag ggctacagagaggcc ctgtgcaagtggatcgacttcacaagggattttctgtccaagtataccaagacaacctct atcgatctgtctagc ctgcggccatcctctcagtataaggacctgggcgagtactatgccgagctgaatcccctg ctgtaccacatcagc ttccagagaatcgccgagaaggagatcatggatgccgtggagacaggcaagctgtacctg ttccagatctataac aaggactttgccaagggccaccacggcaagcctaatctgcacacactgtattggaccggc ctgttttctccagag aacctggccaagacaagcatcaagctgaatggccaggccgagctgttctaccgccctaag tccaggatgaagagg atggcacaccggctgggagagaagatgctgaacaagaagctgaaggatcagaaaacccca atccccgacaccctg taccaggagctgtacgactatgtgaatcacagactgtcccacgacctgtctgatgaggcc agggccctgctgccc aacgtgatcaccaaggaggtgtctcacgagatcatcaaggataggcgctttaccagcgac aagttctttttccac gtgcctatcacactgaactatcaggccgccaattccccatctaagttcaaccagagggtg aatgcctacctgaag gagcaccccgagacacctatcatcggcatcgcccggggcgagagaaacctgatctatatc acagtgatcgactcc accggcaagatcctggagcagcggagcctgaacaccatccagcagtttgattaccagaag aagctggacaacagg gagaaggagagggtggcagcaaggcaggcctggtctgtggtgggcacaatcaaggatctg aagcagggctatctg agccaggtcatccacgagatcgtggacctgatgatccactaccaggccgtggtggtgctg gagaacctgaatttc ggctttaagagcaagaggaccggcatcgccgagaaggccgtgtaccagcagttcgagaag atgctgatcgataag ctgaattgcctggtgctgaaggactatccagcagagaaagtgggaggcgtgctgaaccca taccagctgacagac cagttcacctcctttgccaagatgggcacccagtctggcttcctgttttacgtgcctgcc ccatatacatctaag atcgatcccctgaccggcttcgtggaccccttcgtgtggaaaaccatcaagaatcacgag agccgcaagcacttc ctggagggcttcgactttctgcactacgacgtgaaaaccggcgacttcatcctgcacttt aagatgaacagaaat ctgtccttccagaggggcctgcccggctttatgcctgcatgggatatcgtgttcgagaag aacgagacacagttt gacgccaagggcacccctttcatcgccggcaagagaatcgtgccagtgatcgagaatcac agattcaccggcaga taccgggacctgtatcctgccaacgagctgatcgccctgctggaggagaagggcatcgtg ttcagggatggctcc aacatcctgccaaagctgctggagaatgacgattctcacgccatcgacaccatggtggcc ctgatccgcagcgtg ctgcagatgcggaactccaatgccgccacaggcgaggactatatcaacagccccgtgcgc gatctgaatggcgtg tgcttcgactcccggtttcagaacccagagtggcccatggacgccgatgccaatggcgcc taccacatcgccctg aagggccagctgctgctgaatcacctgaaggagagcaaggatctgaagctgcagaacggc atctccaatcaggac tggctggcctacatccaggagctgcgcaac Exemplary nucleic acid sequence of Construct G: Cpf 1 (D908A) - XTEN - FokI (D450A)- polypeptide linker - Fokll (SEQ ID NO: 37) atgacacagttcgagggctttaccaacctgtatcaggtgagcaagacactgcggtttgag ctgatcccacagggc aagaccctgaagcacatccaggagcagggcttcatcgaggaggacaaggcccgcaatgat cactacaaggagctg aagcccatcatcgatcggatctacaagacctatgccgaccagtgcctgcagctggtgcag ctggattgggagaac ctgagcgccgccatcgactcctatagaaaggagaaaaccgaggagacaaggaacgccctg atcgaggagcaggcc acatatcgcaatgccatccacgactacttcatcggccggacagacaacctgaccgatgcc atcaataagagacac gccgagatctacaagggcctgttcaaggccgagctgtttaatggcaaggtgctgaagcag ctgggcaccgtgacc acaaccgagcacgagaacgccctgctgcggagcttcgacaagtttacaacctacttctcc ggcttttatagaaac aggaagaacgtgttcagcgccgaggatatcagcacagccatcccacaccgcatcgtgcag gacaacttccccaag tttaaggagaattgtcacatcttcacacgcctgatcaccgccgtgcccagcctgcgggag cactttgagaacgtg aagaaggccatcggcatcttcgtgagcacctccatcgaggaggtgttttccttccctttt tataaccagctgctg acacagacccagatcgacctgtataaccagctgctgggaggaatctctcgggaggcaggc accgagaagatcaag ggcctgaacgaggtgctgaatctggccatccagaagaatgatgagacagcccacatcatc gcctccctgccacac agattcatccccctgtttaagcagatcctgtccgataggaacaccctgtctttcatcctg gaggagtttaagagc gacgaggaagtgatccagtccttctgcaagtacaagacactgctgagaaacgagaacgtg ctggagacagccgag gccctgtttaacgagctgaacagcatcgacctgacacacatcttcatcagccacaagaag ctggagacaatcagc agcgccctgtgcgaccactgggatacactgaggaatgccctgtatgagcggagaatctcc gagctgacaggcaag atcaccaagtctgccaaggagaaggtgcagcgcagcctgaagcacgaggatatcaacctg caggagatcatctct gccgcaggcaaggagctgagcgaggccttcaagcagaaaaccagcgagatcctgtcccac gcacacgccgccctg gatcagccactgcctacaaccctgaagaagcaggaggagaaggagatcctgaagtctcag ctggacagcctgctg ggcctgtaccacctgctggactggtttgccgtggatgagtccaacgaggtggaccccgag ttctctgcccggctg accggcatcaagctggagatggagccttctctgagcttctacaacaaggccagaaattat gccaccaagaagccc tactccgtggagaagttcaagctgaactttcagatgcctacactggccagaggctgggac gtgaatgtggagaag aacagaggcgccatcctgtttgtgaagaacggcctgtactatctgggcatcatgccaaag cagaagggcaggtat aaggccctgagcttcgagcccacagagaaaaccagcgagggctttgataagatgtactat gactacttccctgat gccgccaagatgatcccaaagtgcagcacccagctgaaggccgtgacagcccactttcag acccacacaaccccc atcctgctgtccaacaatttcatcgagcctctggagatcacaaaggagatctacgacctg aacaatcctgagaag gagccaaagaagtttcagacagcctacgccaagaaaaccggcgaccagaagggctacaga gaggccctgtgcaag tggatcgacttcacaagggattttctgtccaagtataccaagacaacctctatcgatctg tctagcctgcggcca tcctctcagtataaggacctgggcgagtactatgccgagctgaatcccctgctgtaccac atcagcttccagaga atcgccgagaaggagatcatggatgccgtggagacaggcaagctgtacctgttccagatc tataacaaggacttt gccaagggccaccacggcaagcctaatctgcacacactgtattggaccggcctgttttct ccagagaacctggcc aagacaagcatcaagctgaatggccaggccgagctgttctaccgccctaagtccaggatg aagaggatggcacac cggctgggagagaagatgctgaacaagaagctgaaggatcagaaaaccccaatccccgac accctgtaccaggag ctgtacgactatgtgaatcacagactgtcccacgacctgtctgatgaggccagggccctg ctgcccaacgtgatc accaaggaggtgtctcacgagatcatcaaggataggcgctttaccagcgacaagttcttt ttccacgtgcctatc acactgaactatcaggccgccaattccccatctaagttcaaccagagggtgaatgcctac ctgaaggagcacccc gagacacctatcatcggcatcgcccggggcgagagaaacctgatctatatcacagtgatc gactccaccggcaag atcctggagcagcggagcctgaacaccatccagcagtttgattaccagaagaagctggac aacagggagaaggag agggtggcagcaaggcaggcctggtctgtggtgggcacaatcaaggatctgaagcagggc tatctgagccaggtc atccacgagatcgtggacctgatgatccactaccaggccgtggtggtgctggagaacctg aatttcggctttaag agcaagaggaccggcatcgccgagaaggccgtgtaccagcagttcgagaagatgctgatc gataagctgaattgc ctggtgctgaaggactatccagcagagaaagtgggaggcgtgctgaacccataccagctg acagaccagttcacc tcctttgccaagatgggcacccagtctggcttcctgttttacgtgcctgccccatataca tctaagatcgatccc ctgaccggcttcgtggaccccttcgtgtggaaaaccatcaagaatcacgagagccgcaag cacttcctggagggc ttcgactttctgcactacgacgtgaaaaccggcgacttcatcctgcactttaagatgaac agaaatctgtccttc cagaggggcctgcccggctttatgcctgcatgggatatcgtgttcgagaagaacgagaca cagtttgacgccaag ggcacccctttcatcgccggcaagagaatcgtgccagtgatcgagaatcacagattcacc ggcagataccgggac ctgtatcctgccaacgagctgatcgccctgctggaggagaagggcatcgtgttcagggat ggctccaacatcctg ccaaagctgctggagaatgacgattctcacgccatcgacaccatggtggccctgatccgc agcgtgctgcagatg cggaactccaatgccgccacaggcgaggactatatcaacagccccgtgcgcgatctgaat ggcgtgtgcttcgac tcccggtttcagaacccagagtggcccatggacgccgatgccaatggcgcctaccacatc gccctgaagggccag ctgctgctgaatcacctgaaggagagcaaggatctgaagctgcagaacggcatctccaat caggactggctggcc tacatccaggagctgcgcaacagcggcagcgagactcccgggacctcagagtccgccaca cccgaaagtcaactg gtgaagagcgagctggaagagaagaaaagcgagctcagacataagctgaagtacgttccc cacgaatacattgaa ctgatagaaatcgctagaaacagtacgcaagacagaatactggaaatgaaggtgatggag ttcttcatgaaggtt tacggctatcgtggcaaacacctcgggggctcccggaagcccgccggggctatctacacc gtgggcagtcccatc gactatggcgtgatcgtggacaccaaagcttatagcggcggatataatctccccatcggc caagccgatgagatg cagaggtatgtggaggagaaccaaacaagaaacaagcatatcaaccccaacgagtggtgg aaggtttatcctagc tcggtgaccgagtttaagttcctattcgtgtctggccacttcaagggcaactataaggca cagctcactagactg aatcatatcacgaattgcaacggcgccgtgttatccgtggaggagctactgatcggcgga gagatgatcaaagcc ggcaccctgaccctggaagaggtgagaagaaagtttaacaatggcgaaataaatttcggc agcggaagtggaagc ggctccatcactagaaccaccaaccctagaaacgtggtgcccaagatctacatgagcgcc ggcagcatccccctg accacccacatcaccaactcaattcagcccaccctgtggaccatcggcagcatcaacggc gtggcccccctggcc aagagcatcaagctgggcatccccgtgaccggcagcgcctacaccgatcagaccaccgcc atggtgagaaagaag gtgagcgtgttcatgggcagcggcagcgggagcggctcatcgcagctggttaagagcgag ttagaagaaaaaaag agcgaactgcggcataaactgaagtatgtcccacacgagtacatcgaactgatcgagatc gcgagaaactctacc caagacagaattctggagatgaaagtaatggaatttttcatgaaggtgtatggatataga gggaagcacctgggt ggcagcagaaaacccgacggcgccatctacactgtggggagccccatagactatggtgtg atcgtggataccaag gcgtatagcggcggttacaatctgcccattgggcaagcggacgagatgcaaagatatgtg gaagagaatcagacg aggaacaagcacattaaccctaatgagtggtggaaggtctaccctagctccgttaccgag ttcaagttcctgttt gtgagcgggcattttaagggcaactacaaggcacagctgacccgcctgaaccacataaca aactgcaacggtgcc gtgctgagcgtagaagagttgctaatcggcggcgagatgatcaaggccggcacgctaacc ctcgaagaggtgcgc agaaagttcaataacggcgaaatcaatttc

Exemplary nucleic acid sequence of Construct H: Cpf 1 (D908A) - XTEN - Fokl- polypeptide linker - Fokll (D450A) (SEQ ID NO: 38) atgacacagttcgagggctttaccaacctgtatcaggtgagcaagacactgcggtttgag ctgatcccacagggc aagaccctgaagcacatccaggagcagggcttcatcgaggaggacaaggcccgcaatgat cactacaaggagctg aagcccatcatcgatcggatctacaagacctatgccgaccagtgcctgcagctggtgcag ctggattgggagaac ctgagcgccgccatcgactcctatagaaaggagaaaaccgaggagacaaggaacgccctg atcgaggagcaggcc acatatcgcaatgccatccacgactacttcatcggccggacagacaacctgaccgatgcc atcaataagagacac gccgagatctacaagggcctgttcaaggccgagctgtttaatggcaaggtgctgaagcag ctgggcaccgtgacc acaaccgagcacgagaacgccctgctgcggagcttcgacaagtttacaacctacttctcc ggcttttatagaaac aggaagaacgtgttcagcgccgaggatatcagcacagccatcccacaccgcatcgtgcag gacaacttccccaag tttaaggagaattgtcacatcttcacacgcctgatcaccgccgtgcccagcctgcgggag cactttgagaacgtg aagaaggccatcggcatcttcgtgagcacctccatcgaggaggtgttttccttccctttt tataaccagctgctg acacagacccagatcgacctgtataaccagctgctgggaggaatctctcgggaggcaggc accgagaagatcaag ggcctgaacgaggtgctgaatctggccatccagaagaatgatgagacagcccacatcatc gcctccctgccacac agattcatccccctgtttaagcagatcctgtccgataggaacaccctgtctttcatcctg gaggagtttaagagc gacgaggaagtgatccagtccttctgcaagtacaagacactgctgagaaacgagaacgtg ctggagacagccgag gccctgtttaacgagctgaacagcatcgacctgacacacatcttcatcagccacaagaag ctggagacaatcagc agcgccctgtgcgaccactgggatacactgaggaatgccctgtatgagcggagaatctcc gagctgacaggcaag atcaccaagtctgccaaggagaaggtgcagcgcagcctgaagcacgaggatatcaacctg caggagatcatctct gccgcaggcaaggagctgagcgaggccttcaagcagaaaaccagcgagatcctgtcccac gcacacgccgccctg gatcagccactgcctacaaccctgaagaagcaggaggagaaggagatcctgaagtctcag ctggacagcctgctg ggcctgtaccacctgctggactggtttgccgtggatgagtccaacgaggtggaccccgag ttctctgcccggctg accggcatcaagctggagatggagccttctctgagcttctacaacaaggccagaaattat gccaccaagaagccc tactccgtggagaagttcaagctgaactttcagatgcctacactggccagaggctgggac gtgaatgtggagaag aacagaggcgccatcctgtttgtgaagaacggcctgtactatctgggcatcatgccaaag cagaagggcaggtat aaggccctgagcttcgagcccacagagaaaaccagcgagggctttgataagatgtactat gactacttccctgat gccgccaagatgatcccaaagtgcagcacccagctgaaggccgtgacagcccactttcag acccacacaaccccc atcctgctgtccaacaatttcatcgagcctctggagatcacaaaggagatctacgacctg aacaatcctgagaag gagccaaagaagtttcagacagcctacgccaagaaaaccggcgaccagaagggctacaga gaggccctgtgcaag tggatcgacttcacaagggattttctgtccaagtataccaagacaacctctatcgatctg tctagcctgcggcca tcctctcagtataaggacctgggcgagtactatgccgagctgaatcccctgctgtaccac atcagcttccagaga atcgccgagaaggagatcatggatgccgtggagacaggcaagctgtacctgttccagatc tataacaaggacttt gccaagggccaccacggcaagcctaatctgcacacactgtattggaccggcctgttttct ccagagaacctggcc aagacaagcatcaagctgaatggccaggccgagctgttctaccgccctaagtccaggatg aagaggatggcacac cggctgggagagaagatgctgaacaagaagctgaaggatcagaaaaccccaatccccgac accctgtaccaggag ctgtacgactatgtgaatcacagactgtcccacgacctgtctgatgaggccagggccctg ctgcccaacgtgatc accaaggaggtgtctcacgagatcatcaaggataggcgctttaccagcgacaagttcttt ttccacgtgcctatc acactgaactatcaggccgccaattccccatctaagttcaaccagagggtgaatgcctac ctgaaggagcacccc gagacacctatcatcggcatcgcccggggcgagagaaacctgatctatatcacagtgatc gactccaccggcaag atcctggagcagcggagcctgaacaccatccagcagtttgattaccagaagaagctggac aacagggagaaggag agggtggcagcaaggcaggcctggtctgtggtgggcacaatcaaggatctgaagcagggc tatctgagccaggtc atccacgagatcgtggacctgatgatccactaccaggccgtggtggtgctggagaacctg aatttcggctttaag agcaagaggaccggcatcgccgagaaggccgtgtaccagcagttcgagaagatgctgatc gataagctgaattgc ctggtgctgaaggactatccagcagagaaagtgggaggcgtgctgaacccataccagctg acagaccagttcacc tcctttgccaagatgggcacccagtctggcttcctgttttacgtgcctgccccatataca tctaagatcgatccc ctgaccggcttcgtggaccccttcgtgtggaaaaccatcaagaatcacgagagccgcaag cacttcctggagggc ttcgactttctgcactacgacgtgaaaaccggcgacttcatcctgcactttaagatgaac agaaatctgtccttc cagaggggcctgcccggctttatgcctgcatgggatatcgtgttcgagaagaacgagaca cagtttgacgccaag ggcacccctttcatcgccggcaagagaatcgtgccagtgatcgagaatcacagattcacc ggcagataccgggac ctgtatcctgccaacgagctgatcgccctgctggaggagaagggcatcgtgttcagggat ggctccaacatcctg ccaaagctgctggagaatgacgattctcacgccatcgacaccatggtggccctgatccgc agcgtgctgcagatg cggaactccaatgccgccacaggcgaggactatatcaacagccccgtgcgcgatctgaat ggcgtgtgcttcgac tcccggtttcagaacccagagtggcccatggacgccgatgccaatggcgcctaccacatc gccctgaagggccag ctgctgctgaatcacctgaaggagagcaaggatctgaagctgcagaacggcatctccaat caggactggctggcc tacatccaggagctgcgcaacagcggcagcgagactcccgggacctcagagtccgccaca cccgaaagtcaactg gtgaagagcgagctggaagagaagaaaagcgagctcagacataagctgaagtacgttccc cacgaatacattgaa ctgatagaaatcgctagaaacagtacgcaagacagaatactggaaatgaaggtgatggag ttcttcatgaaggtt tacggctatcgtggcaaacacctcgggggctcccggaagcccgacggggctatctacacc gtgggcagtcccatc gactatggcgtgatcgtggacaccaaagcttatagcggcggatataatctccccatcggc caagccgatgagatg cagaggtatgtggaggagaaccaaacaagaaacaagcatatcaaccccaacgagtggtgg aaggtttatcctagc tcggtgaccgagtttaagttcctattcgtgtctggccacttcaagggcaactataaggca cagctcactagactg aatcatatcacgaattgcaacggcgccgtgttatccgtggaggagctactgatcggcgga gagatgatcaaagcc ggcaccctgaccctggaagaggtgagaagaaagtttaacaatggcgaaataaatttcggc agcggaagtggaagc ggctccatcactagaaccaccaaccctagaaacgtggtgcccaagatctacatgagcgcc ggcagcatccccctg accacccacatcaccaactcaattcagcccaccctgtggaccatcggcagcatcaacggc gtggcccccctggcc aagagcatcaagctgggcatccccgtgaccggcagcgcctacaccgatcagaccaccgcc atggtgagaaagaag gtgagcgtgttcatgggcagcggcagcgggagcggctcatcgcagctggttaagagcgag ttagaagaaaaaaag agcgaactgcggcataaactgaagtatgtcccacacgagtacatcgaactgatcgagatc gcgagaaactctacc caagacagaattctggagatgaaagtaatggaatttttcatgaaggtgtatggatataga gggaagcacctgggt ggcagcagaaaacccgccggcgccatctacactgtggggagccccatagactatggtgtg atcgtggataccaag gcgtatagcggcggttacaatctgcccattgggcaagcggacgagatgcaaagatatgtg gaagagaatcagacg aggaacaagcacattaaccctaatgagtggtggaaggtctaccctagctccgttaccgag ttcaagttcctgttt gtgagcgggcattttaagggcaactacaaggcacagctgacccgcctgaaccacataaca aactgcaacggtgcc gtgctgagcgtagaagagttgctaatcggcggcgagatgatcaaggccggcacgctaacc ctcgaagaggtgcgc agaaagttcaataacggcgaaatcaatttc

Exemplary nucleic acid sequence of Construct I: FokI (D450A) - polypeptide linker - Fokll

- XTEN - Cpfl (D908A) (SEQ ID NO: 39) atgcaactggtgaagagcgagctggaagagaagaaaagcgagctcagacataagctgaag tacgttccccacgaa tacattgaactgatagaaatcgctagaaacagtacgcaagacagaatactggaaatgaag gtgatggagttcttc atgaaggtttacggctatcgtggcaaacacctcgggggctcccggaagcccgccggggct atctacaccgtgggc agtcccatcgactatggcgtgatcgtggacaccaaagcttatagcggcggatataatctc cccatcggccaagcc gatgagatgcagaggtatgtggaggagaaccaaacaagaaacaagcatatcaaccccaac gagtggtggaaggtt tatcctagctcggtgaccgagtttaagttcctattcgtgtctggccacttcaagggcaac tataaggcacagctc actagactgaatcatatcacgaattgcaacggcgccgtgttatccgtggaggagctactg atcggcggagagatg atcaaagccggcaccctgaccctggaagaggtgagaagaaagtttaacaatggcgaaata aatttcggcagcgga agtggaagcggctccatcactagaaccaccaaccctagaaacgtggtgcccaagatctac atgagcgccggcagc atccccctgaccacccacatcaccaactcaattcagcccaccctgtggaccatcggcagc atcaacggcgtggcc cccctggccaagagcatcaagctgggcatccccgtgaccggcagcgcctacaccgatcag accaccgccatggtg agaaagaaggtgagcgtgttcatgggcagcggcagcgggagcggctcatcgcagctggtt aagagcgagttagaa gaaaaaaagagcgaactgcggcataaactgaagtatgtcccacacgagtacatcgaactg atcgagatcgcgaga aactctacccaagacagaattctggagatgaaagtaatggaatttttcatgaaggtgtat ggatatagagggaag cacctgggtggcagcagaaaacccgacggcgccatctacactgtggggagccccatagac tatggtgtgatcgtg gataccaaggcgtatagcggcggttacaatctgcccattgggcaagcggacgagatgcaa agatatgtggaagag aatcagacgaggaacaagcacattaaccctaatgagtggtggaaggtctaccctagctcc gttaccgagttcaag ttcctgtttgtgagcgggcattttaagggcaactacaaggcacagctgacccgcctgaac cacataacaaactgc aacggtgccgtgctgagcgtagaagagttgctaatcggcggcgagatgatcaaggccggc acgctaaccctcgaa gaggtgcgcagaaagttcaataacggcgaaatcaatttcagcggcagcgagactcccggg acctcagagtccgcc acacccgaaagtacacagttcgagggctttaccaacctgtatcaggtgagcaagacactg cggtttgagctgatc ccacagggcaagaccctgaagcacatccaggagcagggcttcatcgaggaggacaaggcc cgcaatgatcactac aaggagctgaagcccatcatcgatcggatctacaagacctatgccgaccagtgcctgcag ctggtgcagctggat tgggagaacctgagcgccgccatcgactcct at agaaaggagaaaaccgaggagacaaggaacgccct gategag gagcaggccacatatcgcaatgccatccacgactacttcatcggccggacagacaacctg accgatgccatcaat aagagacacgccgagatctacaagggcctgttcaaggccgagctgtttaatggcaaggtg ctgaagcagctgggc accgtgaccacaaccgagcacgagaacgccctgctgcggagcttcgacaagtttacaacc tacttctccggcttt tatagaaacaggaagaacgtgttcagcgccgaggatatcagcacagccatcccacaccgc atcgtgcaggacaac ttccccaagtttaaggagaattgtcacatcttcacacgcctgatcaccgccgtgcccagc ctgcgggagcacttt gagaacgtgaagaaggccatcggcatcttcgtgagcacctccatcgaggaggtgttttcc ttccctttttataac cagctgctgacacagacccagatcgacctgtataaccagctgctgggaggaatctctcgg gaggcaggcaccgag aagatcaagggcctgaacgaggtgctgaatctggccatccagaagaatgatgagacagcc cacatcatcgcctcc ctgccacacagattcatccccctgtttaagcagatcctgtccgataggaacaccctgtct ttcatcctggaggag tttaagagcgacgaggaagtgatccagtccttctgcaagtacaagacactgctgagaaac gagaacgtgctggag acagccgaggccctgtttaacgagctgaacagcatcgacctgacacacatcttcatcagc cacaagaagctggag acaatcagcagcgccctgtgcgaccactgggatacactgaggaatgccctgtatgagcgg agaatctccgagctg acaggcaagatcaccaagtctgccaaggagaaggtgcagcgcagcctgaagcacgaggat atcaacctgcaggag atcatctctgccgcaggcaaggagctgagcgaggccttcaagcagaaaaccagcgagatc ctgtcccacgcacac gccgccctggatcagccactgcctacaaccctgaagaagcaggaggagaaggagatcctg aagtctcagctggac agcctgctgggcctgtaccacctgctggactggtttgccgtggatgagtccaacgaggtg gaccccgagttctct gcccggctgaccggcatcaagctggagatggagccttctctgagcttctacaacaaggcc agaaattatgccacc aagaagccctactccgtggagaagttcaagctgaactttcagatgcctacactggccaga ggctgggacgtgaat gtggagaagaacagaggcgccatcctgtttgtgaagaacggcctgtactatctgggcatc atgccaaagcagaag ggcaggtataaggccctgagcttcgagcccacagagaaaaccagcgagggctttgataag atgtactatgactac ttccctgatgccgccaagatgatcccaaagtgcagcacccagctgaaggccgtgacagcc cactttcagacccac acaacccccatcctgctgtccaacaatttcatcgagcctctggagatcacaaaggagatc tacgacctgaacaat cctgagaaggagccaaagaagtttcagacagcctacgccaagaaaaccggcgaccagaag ggctacagagaggcc ctgtgcaagtggatcgacttcacaagggattttctgtccaagtataccaagacaacctct atcgatctgtctagc ctgcggccatcctctcagtataaggacctgggcgagtactatgccgagctgaatcccctg ctgtaccacatcagc ttccagagaatcgccgagaaggagatcatggatgccgtggagacaggcaagctgtacctg ttccagatctataac aaggactttgccaagggccaccacggcaagcctaatctgcacacactgtattggaccggc ctgttttctccagag aacctggccaagacaagcatcaagctgaatggccaggccgagctgttctaccgccctaag tccaggatgaagagg atggcacaccggctgggagagaagatgctgaacaagaagctgaaggatcagaaaacccca atccccgacaccctg taccaggagctgtacgactatgtgaatcacagactgtcccacgacctgtctgatgaggcc agggccctgctgccc aacgtgatcaccaaggaggtgtctcacgagatcatcaaggataggcgctttaccagcgac aagttctttttccac gtgcctatcacactgaactatcaggccgccaattccccatctaagttcaaccagagggtg aatgcctacctgaag gagcaccccgagacacctatcatcggcatcgcccggggcgagagaaacctgatctatatc acagtgatcgactcc accggcaagatcctggagcagcggagcctgaacaccatccagcagtttgattaccagaag aagctggacaacagg gagaaggagagggtggcagcaaggcaggcctggtctgtggtgggcacaatcaaggatctg aagcagggctatctg agccaggtcatccacgagatcgtggacctgatgatccactaccaggccgtggtggtgctg gagaacctgaatttc ggctttaagagcaagaggaccggcatcgccgagaaggccgtgtaccagcagttcgagaag atgctgatcgataag ctgaattgcctggtgctgaaggactatccagcagagaaagtgggaggcgtgctgaaccca taccagctgacagac cagttcacctcctttgccaagatgggcacccagtctggcttcctgttttacgtgcctgcc ccatatacatctaag atcgatcccctgaccggcttcgtggaccccttcgtgtggaaaaccatcaagaatcacgag agccgcaagcacttc ctggagggcttcgactttctgcactacgacgtgaaaaccggcgacttcatcctgcacttt aagatgaacagaaat ctgtccttccagaggggcctgcccggctttatgcctgcatgggatatcgtgttcgagaag aacgagacacagttt gacgccaagggcacccctttcatcgccggcaagagaatcgtgccagtgatcgagaatcac agattcaccggcaga taccgggacctgtatcctgccaacgagctgatcgccctgctggaggagaagggcatcgtg ttcagggatggctcc aacatcctgccaaagctgctggagaatgacgattctcacgccatcgacaccatggtggcc ctgatccgcagcgtg ctgcagatgcggaactccaatgccgccacaggcgaggactatatcaacagccccgtgcgc gatctgaatggcgtg tgcttcgactcccggtttcagaacccagagtggcccatggacgccgatgccaatggcgcc taccacatcgccctg aagggccagctgctgctgaatcacctgaaggagagcaaggatctgaagctgcagaacggc atctccaatcaggac tggctggcctacatccaggagctgcgcaac

The nucleic acids can comprise any isolated or purified nucleotide sequence which encodes any of fusion polypeptides, portions, or functional variants thereof. Alternatively, the nucleotide sequence can comprise a nucleotide sequence which is degenerate to any of the sequences or a combination of degenerate sequences.

Also provided are vectors comprising said nucleic acids. Nucleic acids provided in the present disclosure include nucleic acid sequences which encode proteins, guide RNAs

(gRNAs), and selection cassettes (i.e. ampicillin resistance cassettes and puromycin resistance cassettes), as well as nucleic acid sequences which control the expression of the same, i.e. promoters, enhancers, polyA signals etc.

Nucleic acids provided in the present disclosure include features directed to promoting or controlling replication of said nucleic acids in systems for manufacturing said nucleic acids. In some embodiments, nucleic acids for modifying cells are produced in insect cells, yeast cells, or bacterial cells.

Nucleic acids encoding any of the fusion polyproteins described herein can be incorporated into a vector, such as a recombinant expression vector. As described herein, the terms “recombinant expression vector” and “vector” may be used interchangeably and refer to a genetically-modified oligonucleotide or polynucleotide construct that permits the expression of an mRNA, protein, polypeptide, or peptide by a host cell, when the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with the cell under conditions sufficient to have the mRNA, protein, polypeptide, or peptide expressed within the cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.

In some embodiments, vectors are not naturally-occurring as a whole. However, parts of the vectors can be naturally-occurring. The inventive recombinant expression vectors can comprise any type of nucleotides, including, but not limited to DNA and RNA, which can be single- stranded or double-stranded, synthesized or obtained in part from natural sources, and which can contain natural, non-natural or altered nucleotides. In some embodiments, the vector is a DNA vector. In some embodiments, the vector is an RNA vector. The vectors can comprise naturally-occurring or non-naturally-occurring intemucleotide linkages, or both types of linkages. In some embodiments, a non-naturally occurring or altered nucleotides or intemucleotide linkages do not hinder the transcription or replication of the vector.

The vector may be any suitable recombinant expression vector, and can be used to transform or transfect any suitable host cell. Suitable vectors include those designed for propagation and expansion or for expression or both, such as plasmids and viruses. A vector can be selected from the group consisting of the pUC series (Fermentas Life Sciences, Glen Burnie, MD), the pBluescript series (Stratagene, LaJolla, CA), the pET series (Novagen, Madison, WI), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, CA). Bacteriophage vectors, such as LGT1O, XGT11, LZapII (Stratagene), XEMBT4, and XNMI149, also can be used. Examples of plant expression vectors include pBIOl, pBI101.2, pBI101.3, pBH21 and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-CI, pMAM, and pMAMneo (Clontech). The recombinant expression vector may be a viral vector, e.g., an adenoviral vector, a retroviral vector, or a lentiviral vector.

In some embodiments, the vectors of the invention can be prepared using standard recombinant DNA techniques described in, for example, Green et al., supra. Constructs of expression vectors, which are circular or linear, can be prepared to contain a replication system functional in a prokaryotic or eukaryotic host cell. Replication systems can be derived, e.g., from ColEl, 2p plasmid, X, SV40, bovine papilloma virus, and the like.

A recombinant expression vector may comprise regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host cell (e.g., bacterium, fungus, plant, or animal) into which the vector is to be introduced, as appropriate, and taking into consideration whether the vector is DNA- or RNA-based. A recombinant expression vector may also comprise restriction sites to facilitate cloning.

A vector can include one or more marker genes, which allow for selection of transformed or transfected host cells. Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation in an auxotrophic host to provide prototrophy, and the like. Suitable marker genes for the inventive expression vectors include, for instance, neomycin/G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, puromycin resistance genes, and ampicillin resistance genes.

In some embodiments, a recombinant expression vector can comprise a native or nonnative promoter operably linked to the nucleotide sequence encoding the fusion polypeptide or to the nucleotide sequence which is complementary to or which hybridizes to the nucleotide sequence encoding the fusion polypeptide. The selection of promoters, e.g., strong, weak, inducible, etc, is within the ordinary skill of the artisan. Similarly, the combining of a nucleotide sequence with a promoter is also within the skill of the artisan. The promoter can be a non-viral promoter or a viral promoter, e.g., a cytomegalovirus (CMV) promoter, a SFFV promoter, an EFla promoter, an SV40 promoter, an RSV promoter, a U6 promoter, a beta actin promoter, or a promoter found in the long-terminal repeat of the murine stem cell virus.

Selection of a promoter for a particular type of polymerase may be desired. As will be understood by one of ordinary skill in the art, transcription in eukaryotic cells is typically performed by three types of RNA polymerases, RNA pol I, and RNA pol II, and RNA pol III. See, e.g., Butler et al. Genes & Dev. (2002) 16: 2583-2592. In some embodiments, the vector comprises an RNA pol I promoter. In some embodiments, the vector comprises an RNA pol II promoter. In some embodiments, the vector comprises an RNA pol III promoter.

Examples of RNA pol II promoters include, without limitation, CMV promoter, CAG promoter, CAGGS promoter, ubiquitin promoter, GAPDH promoter, RSV LTR promoter, EFl A promoter, PGK promoter, UbiC promoter, actin promoter, dihydrofolate promoter, B29 promoter, Desmin promoter, Endoglin promoter, FLT-1 promoter, GFPA promoter, and SYN1 promoter. In some embodiments, the vector comprises a CMV promoter.

Examples of RNA pol III promoters include, without limitation, Hl promoter, U6 promoter, 7SK promoter, 7SK1 promoter, 7SK2 promoter, 7SK3 promoter, and U3 promoter. In some embodiments, the vector comprises a U6 promoter.

Further, the vectors can be made to include a suicide gene. As used herein, the term “suicide gene” refers to a gene that causes the cell expressing the suicide gene to die. A suicide gene can be a gene that confers sensitivity to an agent, e.g., a drug, upon the cell in which the gene is expressed, and causes the cell to die when the cell is contacted with or exposed to the agent. Suicide genes are known in the art and include, for example, the Herpes Simplex Virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, and nitroreductase.

In some embodiments, a nucleic acid encoding any of the fusion polypeptides described herein is operably linked to another nucleic acid sequence. As used herein, the term “operably linked” refers to a functional linkage between, for example, a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence (e.g., encoding any of the fusion polypeptides described herein). Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame. The vectors described herein can be designed for transient expression, stable expression, or for both. Alternatively or in addition, the recombinant expression vectors can be made for constitutive expression or for inducible expression.

Any of the vectors described herein may further comprise one or more additional regulatory elements to modulate expression level and/or stability of the fusion polypeptides expressed from said vectors. Examples of additional regulatory elements include, enhancer sequences, polyA termination sequences (e.g., from BGH, SV40), S/MAR elements, and other posttranscriptional and cis-regulatory elements.

In some embodiments, the vector comprises a cis-regulatory element, such as from hepatitis B virus (HPRE) or Woodchuck hepatitis virus, which are though to increase transgene expression by promoting mRNA exportation from the nucleus to the cytoplasm, enhancing 3' end processing and stability. See, e.g., Sun et al. DNA Cell Biol. (2009) 28(5): 233-250. In some embodiments, the vector comprises a Woodchuck hepatitis posttranscriptional regulatory element (WPRE). In some embodiments, the vector comprises a hepatitis posttranscriptional regulatory element (HPRE).

Any of the vectors described herein may also comprise one or more guide RNAs (gRNAs), which may function, for example, to guide any of the fusion polypeptides described herein to a target sequence in the genome of a host cell.

In some examples, the vectors described herein comprise a promoter operably linked to a coding sequence of any of the fusion polypeptides described herein. In some examples, the vectors described herein comprise a promoter operably linked to a coding sequence of any of the fusion polypeptides described herein, linked to one or more additional regulatory elements. An example composition of a vector comprises an RNA pol II promoter operably linked to a coding sequence of any of the fusion polypeptides described herein, linked to one or more additional regulatory elements. In one example, the vector comprises an RNA pol II promoter operably linked to a coding sequence of any of the fusion polypeptides described herein, linked to one or more additional regulatory elements (e.g., HPRE or WPRE).

In some examples, the vectors described herein comprise a first promoter operably linked to a sequence encoding a gRNA, a second promoter operably linked to a coding sequence of any of the fusion polypeptides described herein, linked to one or more additional regulatory elements. An example composition of a vector comprises an RNA pol III promoter operably linked to a sequence encoding a gRNA, an RNA pol II promoter operably linked to a coding sequence of any of the fusion polypeptides described herein, linked to one or more additional regulatory elements. In one example, the vector comprises an RNA pol III promoter operably linked to a sequence encoding a gRNA, an RNA pol II promoter operably linked to a coding sequence of any of the fusion polypeptides described herein, linked to one or more additional regulatory elements (e.g., HPRE or WPRE).

In some examples, the vectors described herein comprise a first promoter operably linked to a coding sequence of any of the fusion polypeptides described herein, linked to one or more additional regulatory elements, and a second promoter operably linked to a sequence encoding a gRNA. An example composition of a vector comprises an RNA pol II promoter operably linked to a coding sequence of any of the fusion polypeptides described herein, linked to one or more additional regulatory elements, and an RNA pol III promoter operably linked to a sequence encoding a gRNA. In one example, the vector comprises an RNA pol II promoter operably linked to a coding sequence of any of the fusion polypeptides described herein, linked to one or more additional regulatory elements (e.g., HPRE or WPRE), and an RNA pol III promoter operably linked to a sequence encoding a gRNA.

In some embodiments, the vector is any of the exemplary vectors set forth by of any one of SEQ ID NOs: 45-53. The present disclosure also provides vectors comprising a nucleic acid sequence that is at least about 70% or more, e.g., about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to any of the vectors described herein, such as any one of SEQ ID NOs: 45-53.

Methods and Systems of Use of the Fusion Polypeptides

Some aspects of this disclosure provide fusion polypeptides, systems, ribonucleoprotein (RNP) complexes, and methods for generating the genetically engineered cells described herein, e.g., genetically engineered cells comprising a modification in their genome, such as a modification that results in a loss of expression or regulation of a protein, or expression of a variant form of a protein.

The present disclosure provides a system for introducing targeted genomic modifications into a cell of interest. In some embodiments, the system comprises any of the fusion polypeptides described herein and at least one guide RNA (gRNA) that directs or targets the fusion polypeptide to a target site (target sequence) in the genome of the cell. In some embodiments, any of the fusion polypeptides described herein are capable of forming and/or maintaining a ribonucleoprotein (RNP) complex with a gRNA and the RNP complex is capable of binding the target sequence in the genome of a cell. In some embodiments, the system further comprises one or more additional gRNAs that direct or target the fusion polypeptide to additional target site(s) (target sequence) in the genome of the cell. In some embodiments, the system comprises a fusion polypeptide comprising a Cpfl domain that lacks nuclease activity and an endonuclease domain that comprises a first DNA- cleavage domain that is capable of forming a dimer with a second DNA-cleavage domain that is present on a separate fusion polypeptide. In such embodiments, the system may further comprise a second fusion polypeptide comprising a Cpfl domain that lacks nuclease activity and a second endonuclease domain comprising the second DNA-cleavage domain. In some embodiments, the method further comprises contacting the cell with a second fusion polypeptide, or nucleic acid encoding the same.

In some embodiments, the first and second steps detailed above occur simultaneously or in close temporal proximity. In some embodiments, all steps detailed above, if taken, occur simultaneously or in close temporal proximity.

In some aspects, the present disclosure provides methods involving contacting a cell with any of the fusion polypeptides described herein and contacting the cell with a gRNA comprising a targeting domain complementary to a first target sequence in the genome of a cell. In some embodiments, the method further comprises contacting the cell with a second comprising a targeting domain complementary to a second target sequence in the genome of a cell wherein the first target sequence and the second target sequence are not the same and the first fusion polypeptide and second fusion polypeptide are not the same.

In some embodiments, the first target sequence and the second target sequence are on different chromosomes of the genome of the cell. In some embodiments, the first target sequence and the second target sequence are on the same chromosome in the genome of the cell. In some embodiments, the first target sequence and the second target sequence are on the same DNA strand of the chromosome. In some embodiments, the first target sequence and the second target sequence are on different DNA strands of the chromosome. In some embodiments, the first target sequence and the second target sequence are separated by 10- 10,000 nucleotides. In some embodiments, the first target sequence and the second target sequence are separated by 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, or 10,000 nucleotides.

The fusion polypeptides and/or gRNAs described herein can be delivered to a cell in any manner suitable. Various suitable methods for the delivery of a system, e.g., comprising an RNP including a fusion polypeptide and gRNA, may include any suitable method such as, electroporation of RNP into a cell, electroporation of mRNA encoding any of the fusion polypeptides and a gRNA into a cell, various protein or nucleic acid transfection methods, and delivery of encoding RNA or DNA via viral vectors, such as, for example, retroviral (e.g., lentiviral) vectors. Any suitable delivery method is embraced by this disclosure, and the disclosure is not limited in this respect.

In some embodiments, a fusion polypeptide/gRNA complex (RNP complex) is formed, e.g., in vitro, and the cell is contacted with the RNP complex, e.g., via electroporation of the RNP complex into the cell. In some embodiments, the cell is contacted with fusion polypeptide and gRNA separately, and the RNP complex is formed within the cell. In some embodiments, the cell is contacted with a nucleic acid, e.g., a DNA or RNA, encoding the fusion polypeptide, and/or with a nucleic acid encoding the gRNA, or both. In some embodiments, the nucleic acid encoding the fusion polypeptide and/or the nucleic acid encoding the gRNA is an mRNA or an mRNA analog.

In some aspects, the present disclosure provides guide RNAs (gRNAs) that are suitable to target any of the fusion polypeptides described herein to a suitable target site in the genome of a cell. The terms “guide RNA” and “gRNA” are used interchangeably herein and refer to a nucleic acid, typically an RNA, that is bound by an RNA-guided nuclease and promotes the specific targeting or homing of the RNA-guided nuclease to a target nucleic acid, e.g., a target site within the genome of a cell. A gRNA typically comprises at least two domains: a “binding domain,” also sometimes referred to as “gRNA scaffold” or “gRNA backbone” that mediates binding to an RNA-guided nuclease (also referred to as the “binding domain”), and a “targeting domain” that mediates the targeting of the gRNA-bound RNA- guided nuclease to a target site. Some gRNAs comprise additional domains, e.g., complementarity domains, or stem- loop domains. The structures and sequences of naturally occurring gRNA binding domains and engineered variants thereof are well known to those of skill in the art.

Suitable gRNAs for use with CRISPR/Cas nucleases, such as Cpfl nucleases, typically comprise a single RNA molecule, as the naturally occurring Cpfl guide RNA comprises a single RNA molecule. A suitable gRNA may thus be unimolecular (having a single RNA molecule), sometimes referred to herein as single guide RNAs (sgRNAs), or modular (comprising more than one, and typically two, separate RNA molecules). Some exemplary suitable Cpfl gRNA scaffold sequences are provided herein, and additional suitable gRNA scaffold sequences will be apparent to the skilled artisan based on the present disclosure. In some embodiments, e.g., in some embodiments where a Cpfl nuclease is used, a gRNA, may comprise, from 5' to 3': a CRISPR RNA (crRNA) sequence for a CRISPR/Cas nuclease, containing: a proximal domain; a first complementarity domain; a linking domain; and a second complementarity domain (which is complementary to the first complementarity domain); and a targeting domain corresponding to a target site sequence.

Some exemplary suitable Cpfl gRNA scaffold sequences are provided herein, and additional suitable gRNA scaffold sequences will be apparent to the skilled artisan based on the present disclosure. Such additional suitable scaffold sequences include, without limitation, those recited in Jinek, et al. Science (2012) 337(6096):816-821, Ran, et al. Nature Protocols (2013) 8:2281-2308, PCT Publication No. WO 2014/093694, and PCT Publication No. WO 2013/176772, incorporate by reference in their entirety.

A gRNA as provided herein typically comprises a targeting domain that binds to a target site in the genome of a cell. The target site is typically a double-stranded DNA sequence comprising the PAM sequence and, on the same strand as, and directly adjacent to, the PAM sequence, the target domain. The targeting domain of the gRNA typically comprises an RNA sequence that corresponds to the target domain sequence in that it resembles the sequence of the target domain, sometimes with one or more mismatches, but typically comprises an RNA instead of a DNA sequence. The targeting domain of the gRNA thus base-pairs (in full or partial complementarity) with the sequence of the double- stranded target site that is complementary to the sequence of the target domain, and thus with the strand complementary to the strand that comprises the PAM sequence. It will be understood that the targeting domain of the gRNA typically does not include the PAM sequence. It will further be understood that the location of the PAM may be 5’ or 3’ of the target domain sequence, depending on the nuclease employed. For example, the PAM is typically 3’ of the target domain sequences for Cas9 nucleases, and 5’ of the target domain sequence for Casl2a nucleases. For an illustration of the location of the PAM and the mechanism of gRNA binding a target site, see, e.g., Figure 1 of Vanegas et al., Fungal Biol Biotechnol. 2019; 6: 6, which is incorporated by reference herein. For additional illustration and description of the mechanism of gRNA targeting an RNA-guided nuclease to a target site, see Fu Y et al, Nat Biotechnol (2014) (doi: 10.1038/nbt.2808) and Sternberg SH et al., Nature (2014) (doi: 10.1038/naturel3011), both incorporated herein by reference.

The targeting domain may comprise a nucleotide sequence that corresponds to the sequence of the target domain, i.e., the DNA sequence directly adjacent to the PAM sequence (e.g., 5’ of the PAM sequence for Cas9 nucleases, or 3’ of the PAM sequence for Casl2a nucleases). The targeting domain sequence typically comprises between 17 and 30 nucleotides and corresponds fully with the target domain sequence ( i.e., without any mismatch nucleotides), or may comprise one or more, but typically not more than 4, mismatches. As the targeting domain is part of an RNA molecule, the gRNA, it will typically comprise ribonucleotides, while the DNA targeting domain will comprise deoxyribonucleotides .

The structure of a typical Casl2a gRNA can be found, for example in Figure 1 of Zetsche et al. Cell (2015) 163(3): 759-771, which is incorporated by reference herein in its entirety. An exemplary illustration of a Casl2a target site, comprising a 22 nucleotide target domain, and a TTN PAM sequence, as well as of a gRNA comprising a targeting domain that fully corresponds to the target domain (and thus base-pairs with full complementarity with the DNA strand complementary to the strand comprising the target domain and PAM) is provided below:

[ PAM ] [ target domain ( DNA) ]

5 ' -T-T-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N- 3 ' ( DNA )

3 ' -A-A-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-5 ' ( DNA)

I I I I I I I I I I I I I I I I I I I I I I

5 ' - [ gRNA scaffold] -N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-N-3 ' ( RNA) [binding domain ] [ target ing domain ( RNA) ]

In some embodiments, the Casl2a PAM sequence is 5’-T-T-T-V-3’. In some embodiments, the Casl2a PAM sequence is 5’-T-T-V-3’.

While not wishing to be bound by theory, at least in some embodiments, it is believed that the length and complementarity of the targeting domain with the target sequence contributes to specificity of the interaction of the gRNA/Cas molecule complex with a target nucleic acid. In some embodiments, the targeting domain of a gRNA provided herein is 5 to 50 nucleotides in length. In some embodiments, the targeting domain is 15 to 25 nucleotides in length. In some embodiments, the targeting domain is 18 to 22 nucleotides in length. In some embodiments, the targeting domain is 19-21 nucleotides in length. In some embodiments, the targeting domain is 15 nucleotides in length. In some embodiments, the targeting domain is 16 nucleotides in length. In some embodiments, the targeting domain is 17 nucleotides in length. In some embodiments, the targeting domain is 18 nucleotides in length. In some embodiments, the targeting domain is 19 nucleotides in length. In some embodiments, the targeting domain is 20 nucleotides in length. In some embodiments, the targeting domain is 21 nucleotides in length. In some embodiments, the targeting domain is 22 nucleotides in length. In some embodiments, the targeting domain is 23 nucleotides in length. In some embodiments, the targeting domain is 24 nucleotides in length. In some embodiments, the targeting domain is 25 nucleotides in length. In some embodiments, the targeting domain fully corresponds, without mismatch, to a target domain sequence provided herein, or a part thereof. In some embodiments, the targeting domain of a gRNA provided herein comprises 1 mismatch relative to a target domain sequence provided herein. In some embodiments, the targeting domain comprises 2 mismatches relative to the target domain sequence. In some embodiments, the target domain comprises 3 mismatches relative to the target domain sequence.

In some embodiments, a targeting domain comprises a core domain and a secondary targeting domain, e.g., as described in PCT Publication No. WO 2015/157070, which is incorporated by reference in its entirety. In some embodiments, the core domain comprises about 8 to about 13 nucleotides from the 3' end of the targeting domain (e.g., the most 3' 8 to 13 nucleotides of the targeting domain). In some embodiments, the secondary domain is positioned 5' to the core domain. In some embodiments, the core domain corresponds fully with the target domain sequence, or a part thereof. In other embodiments, the core domain may comprise one or more nucleotides that are mismatched with the corresponding nucleotide of the target domain sequence.

The sequence and placement of the above-mentioned domains are described in more detail in PCT Publication No. WO 2015/157070, which is herein incorporated by reference in its entirety, including p. 88-112 therein.

A linking domain may serve to link the first complementarity domain with the second complementarity domain of a unimolecular gRNA. The linking domain can link the first and second complementarity domains covalently or non-covalently. In some embodiments, the linkage is covalent. In some embodiments, the linking domain is, or comprises, a covalent bond interposed between the first complementarity domain and the second complementarity domain. In some embodiments, the linking domain comprises one or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In some embodiments, the linking domain comprises at least one non-nucleotide bond, e.g., as disclosed in PCT Publication No. WO 2018/126176, the entire contents of which are incorporated herein by reference.

In some embodiments, the second complementarity domain of the targeting domain is complementary, at least in part, with the first complementarity domain, and in an embodiment, has sufficient complementarity to the second complementarity domain to form a duplexed region under at least some physiological conditions. In some embodiments, the second complementarity domain can include a sequence that lacks complementarity with the first complementarity domain, e.g., a sequence that loops out from the duplexed region. In some embodiments, the second complementarity domain is 5 to 27 nucleotides in length. In some embodiments, the second complementarity domain is longer than the first complementarity region. In an embodiment, the complementary domain is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. In some embodiments, the second complementarity domain comprises 3 subdomains, which, in the 5' to 3' direction are: a 5' subdomain, a central subdomain, and a 3' subdomain. In some embodiments, the 5' subdomain is 3 to 25, e.g., 4 to 22, 4 to 18, or 4 to 10, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the central subdomain is 1, 2, 3, 4 or 5, e.g., 3, nucleotides in length. In some embodiments, the 3' subdomain is 4 to 9, e.g., 4, 5, 6, 7, 8 or 9 nucleotides in length. In some embodiments, the 5' subdomain and the 3' subdomain of the first complementarity domain, are respectively, complementary, e.g., fully complementary, with the 3' subdomain and the 5' subdomain of the second complementarity domain.

In some embodiments, a gRNA may comprise one or more nucleotides that are chemically modified. Chemical modifications of gRNAs have previously been described, and suitable chemical modifications include any modifications that are beneficial for gRNA function and do not measurably increase any undesired characteristics, e.g., off-target effects, of a given gRNA. Suitable chemical modifications include, for example, those that make a gRNA less susceptible to endo- or exonuclease catalytic activity, and include, without limitation, phosphoro thioate backbone modifications, 2'-O-Me-modifications (e.g., at one or both of the 3’ and 5’ termini), 2’F-modifications, replacement of the ribose sugar with the bicyclic nucleotide-cEt, 3 'thioPACE (MSP) modifications, or any combination thereof. Additional suitable gRNA modifications will be apparent to the skilled artisan based on this disclosure, and such suitable gRNA modifications include, without limitation, those described, e.g., in Rahdar et al. PNAS (2015) 112 (51) E7110-E7117 and Hendel et al., Nat Biotechnol. (2015); 33(9): 985-989, each of which is incorporated herein by reference in its entirety.

For example, a gRNA provided herein may comprise one or more 2’-0 modified nucleotide, e.g., a 2’-O-methyl nucleotide. In some embodiments, the gRNA comprises a 2’- O modified nucleotide, e.g., 2’-O-methyl nucleotide at the 5’ end of the gRNA. In some embodiments, the gRNA comprises a 2’-0 modified nucleotide, e.g., 2’-O-methyl nucleotide at the 3’ end of the gRNA. In some embodiments, the gRNA comprises a 2’-O-modified nucleotide, e.g., a 2’-O-methyl nucleotide at both the 5’ and 3’ ends of the gRNA. In some embodiments, the gRNA is 2’-O-modified, e.g. 2’-O-methyl-modified at the nucleotide at the 5’ end of the gRNA, the second nucleotide from the 5’ end of the gRNA, and the third nucleotide from the 5’ end of the gRNA. In some embodiments, the gRNA is 2’-O-modified, e.g., 2’-O-methyl-modified at the nucleotide at the 3’ end of the gRNA, the second nucleotide from the 3’ end of the gRNA, and the third nucleotide from the 3’ end of the gRNA. In some embodiments, the gRNA is 2’-O-modified, e.g., 2’-O-methyl-modified at the nucleotide at the 5’ end of the gRNA, the second nucleotide from the 5’ end of the gRNA, the third nucleotide from the 5’ end of the gRNA, the nucleotide at the 3’ end of the gRNA, the second nucleotide from the 3’ end of the gRNA, and the third nucleotide from the 3’ end of the gRNA. In some embodiments, the gRNA is 2’-O-modified, e.g., 2’-O-methyl-modified at the second nucleotide from the 3’ end of the gRNA, the third nucleotide from the 3’ end of the gRNA, and at the fourth nucleotide from the 3’ end of the gRNA. In some embodiments, the nucleotide at the 3’ end of the gRNA is not chemically modified. In some embodiments, the nucleotide at the 3’ end of the gRNA does not have a chemically modified sugar. In some embodiments, the gRNA is 2’-O-modified, e.g., 2’-O-methyl-modified, at the nucleotide at the 5’ end of the gRNA, the second nucleotide from the 5’ end of the gRNA, the third nucleotide from the 5’ end of the gRNA, the second nucleotide from the 3’ end of the gRNA, the third nucleotide from the 3’ end of the gRNA, and the fourth nucleotide from the 3’ end of the gRNA. In some embodiments, the 2’-O-methyl nucleotide comprises a phosphate linkage to an adjacent nucleotide. In some embodiments, the 2’-O-methyl nucleotide comprises a phosphorothioate linkage to an adjacent nucleotide. In some embodiments, the 2’-O-methyl nucleotide comprises a thioPACE linkage to an adjacent nucleotide.

In some embodiments, a gRNA provided herein may comprise one or more 2’-O- modified and 3 ’phosphorous -modified nucleotide, e.g., a 2’-O-methyl 3 ’phosphorothioate nucleotide. In some embodiments, the gRNA comprises a 2’-O-modified and 3’phosphorous-modified, e.g., 2’-O-methyl 3 ’phosphorothioate nucleotide at the 5’ end of the gRNA. In some embodiments, the gRNA comprises a 2’-O-modified and 3’phosphorous- modified, e.g., 2’-O-methyl 3’phosphorothioate nucleotide at the 3’ end of the gRNA. In some embodiments, the gRNA comprises a 2’-O-modified and 3’phosphorous-modified, e.g., 2’-O-methyl 3’phosphorothioate nucleotide at the 5’ and 3’ ends of the gRNA. In some embodiments, the gRNA comprises a backbone in which one or more non-bridging oxygen atoms has been replaced with a sulfur atom. In some embodiments, the gRNA is 2’-O- modified and 3’phosphorous-modified, e.g., 2’-O-methyl 3’phosphorothioate-modified at the nucleotide at the 5’ end of the gRNA, the second nucleotide from the 5’ end of the gRNA, and the third nucleotide from the 5’ end of the gRNA. In some embodiments, the gRNA is 2’-O-modified and 3’phosphorous-modified, e.g., 2’-O-methyl 3’phosphorothioate-modified at the nucleotide at the 3’ end of the gRNA, the second nucleotide from the 3’ end of the gRNA, and the third nucleotide from the 3’ end of the gRNA. In some embodiments, the gRNA is 2’-O-modified and 3’phosphorous-modified, e.g., 2’-O-methyl 3’phosphorothioate- modified at the nucleotide at the 5’ end of the gRNA, the second nucleotide from the 5’ end of the gRNA, the third nucleotide from the 5’ end of the gRNA, the nucleotide at the 3’ end of the gRNA, the second nucleotide from the 3’ end of the gRNA, and the third nucleotide from the 3’ end of the gRNA. In some embodiments, the gRNA is 2’-O-modified and 3’phosphorous-modified, e.g., 2’-O-methyl 3’phosphorothioate-modified at the second nucleotide from the 3’ end of the gRNA, the third nucleotide from the 3’ end of the gRNA, and the fourth nucleotide from the 3’ end of the gRNA. In some embodiments, the nucleotide at the 3’ end of the gRNA is not chemically modified. In some embodiments, the nucleotide at the 3’ end of the gRNA does not have a chemically modified sugar. In some embodiments, the gRNA is 2’-O-modified and 3’phosphorous-modified, e.g., 2’-O-methyl 3’phosphorothioate-modified at the nucleotide at the 5’ end of the gRNA, the second nucleotide from the 5’ end of the gRNA, the third nucleotide from the 5’ end of the gRNA, the second nucleotide from the 3’ end of the gRNA, the third nucleotide from the 3’ end of the gRNA, and the fourth nucleotide from the 3’ end of the gRNA.

In some embodiments, a gRNA provided herein may comprise one or more 2’-O- modified and 3’-phosphorous-modified, e.g., 2’-O-methyl 3’thioPACE nucleotide. In some embodiments, the gRNA comprises a 2’-O-modified and 3’phosphorous-modified, e.g., 2’-O- methyl 3’thioPACE nucleotide at the 5’ end of the gRNA. In some embodiments, the gRNA comprises a 2’-O-modified and 3’phosphorous-modified, e.g., 2’-O-methyl 3’thioPACE nucleotide at the 3’ end of the gRNA. In some embodiments, the gRNA comprises a 2’-O- modified and 3’phosphorous-modified, e.g., 2’-O-methyl 3’thioPACE nucleotide at the 5’ and 3’ ends of the gRNA. In some embodiments, the gRNA comprises a backbone in which one or more non-bridging oxygen atoms have been replaced with a sulfur atom and one or more non-bridging oxygen atoms have been replaced with an acetate group. In some embodiments, the gRNA is 2’-O-modified and 3’phosphorous-modified, e.g., 2’-O-methyl 3’ thioPACE-modified at the nucleotide at the 5’ end of the gRNA, the second nucleotide from the 5’ end of the gRNA, and the third nucleotide from the 5’ end of the gRNA. In some embodiments, the gRNA is 2’-O-modified and 3’phosphorous-modified, e.g., 2’-O-methyl 3 ’thioPACE-modified at the nucleotide at the 3’ end of the gRNA, the second nucleotide from the 3’ end of the gRNA, and the third nucleotide from the 3’ end of the gRNA. In some embodiments, the gRNA is 2’-O-modified and 3’phosphorous-modified, e.g., 2’-O-methyl 3 ’thioPACE-modified at the nucleotide at the 5’ end of the gRNA, the second nucleotide from the 5’ end of the gRNA, the third nucleotide from the 5’ end of the gRNA, the nucleotide at the 3’ end of the gRNA, the second nucleotide from the 3’ end of the gRNA, and the third nucleotide from the 3’ end of the gRNA. In some embodiments, the gRNA is 2’-O-modified and 3’phosphorous-modified, e.g., 2’-O-methyl 3 ’thioPACE-modified at the second nucleotide from the 3’ end of the gRNA, the third nucleotide from the 3’ end of the gRNA, and the fourth nucleotide from the 3’ end of the gRNA. In some embodiments, the nucleotide at the 3’ end of the gRNA is not chemically modified. In some embodiments, the nucleotide at the 3’ end of the gRNA does not have a chemically modified sugar. In some embodiments, the gRNA is 2’-O-modified and 3’phosphorous-modified, e.g. 2’-O-methyl 3 ’thioPACE-modified at the nucleotide at the 5’ end of the gRNA, the second nucleotide from the 5’ end of the gRNA, the third nucleotide from the 5’ end of the gRNA, the second nucleotide from the 3’ end of the gRNA, the third nucleotide from the 3’ end of the gRNA, and the fourth nucleotide from the 3’ end of the gRNA.

In some embodiments, a gRNA provided herein comprises a chemically modified backbone. In some embodiments, the gRNA comprises a phosphorothioate linkage. In some embodiments, one or more non-bridging oxygen atoms have been replaced with a sulfur atom. In some embodiments, the nucleotide at the 5’ end of the gRNA, the second nucleotide from the 5’ end of the gRNA, and the third nucleotide from the 5’ end of the gRNA each comprise a phosphorothioate linkage. In some embodiments, the nucleotide at the 3’ end of the gRNA, the second nucleotide from the 3’ end of the gRNA, and the third nucleotide from the 3’ end of the gRNA each comprise a phosphorothioate linkage. In some embodiments, the nucleotide at the 5’ end of the gRNA, the second nucleotide from the 5’ end of the gRNA, the third nucleotide from the 5’ end of the gRNA, the nucleotide at the 3’ end of the gRNA, the second nucleotide from the 3’ end of the gRNA, and the third nucleotide from the 3’ end of the gRNA each comprise a phosphorothioate linkage. In some embodiments, the second nucleotide from the 3’ end of the gRNA, the third nucleotide from the 3’ end of the gRNA, and at the fourth nucleotide from the 3’ end of the gRNA each comprise a phosphorothioate linkage. In some embodiments, the nucleotide at the 5’ end of the gRNA, the second nucleotide from the 5’ end of the gRNA, the third nucleotide from the 5’ end, the second nucleotide from the 3’ end of the gRNA, the third nucleotide from the 3’ end of the gRNA, and the fourth nucleotide from the 3’ end of the gRNA each comprise a phosphorothioate linkage.

In some embodiments, a gRNA provided herein comprises a thioPACE linkage. In some embodiments, the gRNA comprises a backbone in which one or more non-bridging oxygen atoms have been replaced with a sulfur atom and one or more non-bridging oxygen atoms have been replaced with an acetate group. In some embodiments, the nucleotide at the 5’ end of the gRNA, the second nucleotide from the 5’ end of the gRNA, and the third nucleotide from the 5’ end of the gRNA each comprise a thioPACE linkage. In some embodiments, the nucleotide at the 3’ end of the gRNA, the second nucleotide from the 3’ end of the gRNA, and the third nucleotide from the 3’ end of the gRNA each comprise a thioPACE linkage. In some embodiments, the nucleotide at the 5’ end of the gRNA, the second nucleotide from the 5’ end of the gRNA, the third nucleotide from the 5’ end of the gRNA, the nucleotide at the 3’ end of the gRNA, the second nucleotide from the 3’ end of the gRNA, and the third nucleotide from the 3’ end of the gRNA each comprise a thioPACE linkage. In some embodiments, the second nucleotide from the 3’ end of the gRNA, the third nucleotide from the 3’ end of the gRNA, and at the fourth nucleotide from the 3’ end of the gRNA each comprise a thioPACE linkage. In some embodiments, the nucleotide at the 5’ end of the gRNA, the second nucleotide from the 5’ end of the gRNA, the third nucleotide from the 5’ end, the second nucleotide from the 3’ end of the gRNA, the third nucleotide from the 3’ end of the gRNA, and the fourth nucleotide from the 3’ end of the gRNA each comprise a thioPACE linkage.

In some embodiments, a gRNA described herein comprises one or more 2'-O-methyl- 3 '-phosphorothioate nucleotides, e.g., at least 1, 2, 3, 4, 5, or 6 2'-O-methyl-3'- phosphorothioate nucleotides. In some embodiments, a gRNA described herein comprises modified nucleotides (e.g., 2'-O-methyl-3 '-phosphorothioate nucleotides) at one or more of the three terminal positions and the 5’ end and/or at one or more of the three terminal positions and the 3’ end. In some embodiments, the nucleotide at the 5’ end of the gRNA, the second nucleotide from the 5’ end of the gRNA, the third nucleotide from the 5’ end, the second nucleotide from the 3’ end of the gRNA, the third nucleotide from the 3’ end of the gRNA, and the fourth nucleotide from the 3’ end of the gRNA each comprise a 2'-O-methyl- 3'-phosphorothioate nucleotides. In some embodiments, the gRNA may comprise one or more modified nucleotides, e.g., as described in PCT Publication Nos. WO 2017/214460, WO 2016/089433, and WO 2016/164356, which are incorporated by reference their entirety.

The gRNAs provided herein can be delivered to a cell in any manner suitable.

Various suitable methods for the delivery of CRISPR/Cas systems, e.g., comprising an RNP including a gRNA bound to any of the fusion polypeptides described herein, have been described, and exemplary suitable methods include, without limitation, electroporation of a RNP into a cell, electroporation of mRNA encoding any of the fusion polypeptides described herein and a gRNA into a cell, various protein or nucleic acid transfection methods, and delivery of encoding RNA or DNA via viral vectors, such as, for example, retroviral e.g., lentiviral) vectors. Any suitable delivery method is embraced by this disclosure, and the disclosure is not limited in this respect.

Cells

The fusion polypeptides, methods, and strategies provided herein may be applied to any cell or cell type capable of being genetically engineered using the fusion polypeptides and methods described herein. The skilled artisan will understand, however, that the provision of such examples is for the purpose of illustrating some specific embodiments, and additional suitable cells and cell types will be apparent to the skilled artisan based on the present disclosure, which is not limited in this respect.

In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell, yeast cell, fungal cell, or plant cell. In some embodiments, the cell is a mammalian cell, such as a non-human primate cell, a rodent (e.g., mouse or rat) cell, a bovine cell, a porcine cell, an equine cell, or a cell of a domestic animal. In some embodiments, the cell is a human cell or a mouse cell. In some embodiments, the cells may be obtained from a subject, such as a human subject (e.g., a healthy human subject or a human subject having a disease).

In some embodiments, the cells are hematopoietic cells, e.g., hematopoietic stem cells (HSC) or hematopoietic progenitor cells (HPC). In some embodiments, the cells provided herein are hematopoietic stem or progenitor cells. Hematopoietic stem cells (HSCs) are typically capable of giving rise to both myeloid and lymphoid progenitor cells that further give rise to myeloid cells (e.g., monocytes, macrophages, neutrophils, basophils, dendritic cells, erythrocytes, platelets, etc.) and lymphoid cells (e.g., T cells, B cells, NK cells), respectively. HSCs are characterized by the expression off one or more cell surface markers, such as CD34 (e.g., CD34+), which can be used for the identification and/or isolation of HSCs, and absence of cell surface markers associated with commitment to a cell lineage. In some embodiments, the HSCs are peripheral blood HSCs. Methods of obtaining cells, such as hematopoietic stem cells are described, e.g., in PCT Application No.

PCT/US2016/057339, which is herein incorporated by reference in its entirety.

In some embodiments, the cells provided herein are immune effector cells. In some embodiments, the immune effector cell is a lymphocyte. In some embodiments, the immune effector cell is a T-lymphocyte. In some embodiments, the T-lymphocyte is an alpha/beta T- lymphocyte. In some embodiments, the T-lymphocyte is a gamma/delta T-lymphocyte. In some embodiments, the immune effector cell is a natural killer T (NKT cell). In some embodiments, the immune effector cell is a natural killer (NK) cell.

In some embodiments, the cell is a stem cell. In some embodiments, the stem cell is selected from the group consisting of an embryonic stem cell (ESC), an induced pluripotent stem cell (iPSC), a mesenchymal stem cell, or a tissue- specific stem cell.

In some embodiments, a genetically engineered cell provided herein comprises only one genomic modification, e.g., a genomic modification that results in a loss of expression of a protein, for example a protein encoded by or regulated by the target site sequence, or expression of a variant form of the protein. It will be understood that the gene editing methods provided herein may result in genomic modifications in one or both alleles of a target genetic loci. In some embodiments, genetically engineered cells comprising a genomic modification in both alleles of a given genetic locus are preferred.

In some embodiments, a genetically engineered cell provided herein comprises two or more genomic modifications. For example, a population of genetically engineered cells can comprise a plurality of different mutations.

As will be evident to one of ordinary skill in the art, the fusion polypeptides and methods described herein may be used to modify any genetic locus in a cell, including for example protein-coding, non-protein coding, chromosomal, and extra-chromosomal sequences. Accordingly, targeting domains of the gRNAs may be designed to target any genetic locus (i.e., a target site sequence), such as a target site sequence adjacent to a PAM sequence for a corresponding CRISPR/Cas nuclease. In some embodiments, the targeting domain of a gRNA for use with the fusion polypeptides described herein targets a cell surface protein, such as a Type 0, Type 1, or Type 2 cell surface protein. In some embodiments, the targeting domain targets BCMA, CD19, CD20, CD30, ROR1, B7H6, B7H3, CD23, CD33, CD38, C-type lectin like molecule-1 (CLL-1), CS1, EMR2, IL-5, Ll-CAM, PSCA, PSMA, CD138, CD133, CD70, CD5, CD6, CD7, CD13, NKG2D, NKG2D ligand, CLEC12A, CD11, CD117, CD123, CD56, CD34, CD 14, CD66b, CD41, CD61, CD62, CD235a, CD 146, CD326, LMP2, CD22, CD52, CD 10, CD3/TCR, CD79/BCR, and/or CD26.

In some embodiments, the targeting domain of a gRNA for use with the fusion polypeptides described herein targets a cell surface protein associated with a neoplastic or malignant disease or disorder, e.g., with a specific type of cancer, such as, without limitation, CD20, CD22 (Non-Hodgkin's lymphoma, B-cell lymphoma, chronic lymphocytic leukemia (CLL)), CD52 (B-cell CLL), CD33 (Acute myelogenous leukemia (AML)), CD10 (gplOO) (Common (pre-B) acute lymphocytic leukemia and malignant melanoma), CD3/T-cell receptor (TCR) (T-cell lymphoma and leukemia), CD79/B-cell receptor (BCR) (B-cell lymphoma and leukemia), CD26 (epithelial and lymphoid malignancies), human leukocyte antigen (HLA)-DR, HLA-DP, and HLA-DQ (lymphoid malignancies), RCAS1 (gynecological carcinomas, biliary adenocarcinomas and ductal adenocarcinomas of the pancreas) as well as prostate specific membrane antigen.

Additional non-limiting examples of cell surface proteins include CD la, CD lb, CDlc, CDld, CDle, CD2, CD3, CD3d, CD3e, CD3g, CD4, CD5, CD6, CD7, CD8a, CD8b, CD9, CD10, CDl la, CDl lb, CDl lc, CDl ld, CDwl2, CD13, CD14, CD15, CD16, CD16b, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32a, CD32b, CD32c, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD45, CD45RA, CD45RB, CD45RC, CD45RO, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60a, CD61, CD62E, CD62L, CD62P, CD63, CD64a, CD65, CD65s, CD66a, CD66b, CD66c, CD66F, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD75S, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85A, CD85C, CD85D, CD85E, CD85F, CD85G, CD85H, CD85I, CD85J, CD85K, CD86, CD87, CD88, CD89, CD90, CD91, CD92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD99R, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107a, CD107b, CD108, CD109, CD110, CD111, CD112, CD113, CD114, CD115, CD116, CD117, CD118, CD119, CD120a, CD120b, CD121a, CD121b, CD121a, CD121b, CD122, CD123, CD124, CD125, CD126, CD127, CD129, CD130, CD131, CD132, CD133, CD134, CD135, CD136, CD137, CD138, CD139, CD140a, CD140b, CD141, CD142, CD143, CD14, CDwl45, CD146, CD147, CD148, CD150, CD152, CD152, CD153, CD154, CD155, CD156a, CD156b, CD156c, CD157, CD158M, CD158b2, CD158d, CD158el/e2, CD158f, CD158g, CD158h, CD158i, CD158j, CD158k, CD159a, CD159c, CD160, CD161, CD163, CD164, CD165, CD166, CD167a, CD168, CD169, CD170, CD171, CD172a, CD172b, CD172g, CD173, CD174, CD175, CD175s, CD176, CD177, CD178, CD179a, CD179b, CD180, CD181, CD182, CD183, CD184, CD185, CD186, CD191, CD192, CD193, CD194, CD195, CD196, CD197, CDwl98, CDwl99, CD200, CD201, CD202b, CD203c, CD204, CD205, CD206, CD207, CD208, CD209, CD210a, CDw210b, CD212, CD213al, CD213a2, CD215, CD217, CD218a, CD218b, CD220, CD221, CD222, CD223, CD224, CD225, CD226, CD227, CD228, CD229, CD230, CD231, CD232, CD233, CD234, CD235a, CD235b, CD236, CD236R, CD238, CD239, CD240, CD241, CD242, CD243, CD244, CD245, CD246, CD247, CD248, CD249, CD252, CD253, CD254, CD256, CD257, CD258, CD261, CD262, CD263, CD264, CD265, CD266, CD267, CD268, CD269, CD270, CD272, CD272, CD273, CD274, CD275, CD276, CD277, CD278, CD279, CD280, CD281, CD282, CD283, CD284, CD286, CD288, CD289, CD290, CD292, CDw293, CD294, CD295, CD296, CD297, CD298, CD299, CD300a, CD300c, CD300e, CD301, CD302, CD3O3, CD304, CD305, CD306, CD307a, CD307b, CD307c, CD307d, CD307e, CD309, CD312, CD314, CD315, CD316, CD317, CD318, CD319, CD320, CD321, CD322, CD324, CD325, CD326, CD327, CD328, CD329, CD331, CD332, CD333, CD334, CD335, CD336, CD337, CD338, CD339, CD340, CD344, CD349, CD350, CD351, CD352, CD353, CD354, CD355, CD357, CD358, CD359, CD360, CD361, CD362, CD363, CD364, CD365, CD366, CD367, CD368, CD369, CD370, or CD371. See also examples of lineage- specific cell-surface antigens from BD Biosciences Human CD Marker Chart: bdbiosciences.com/content/dam/bdb/campaigns/reagent- education/B D_Reagents_CDMarkerHuman_Poster.pdf (incorporated by reference in its entirety).

Methods of administration to subjects in need thereof

Some aspects of this disclosure provide methods comprising administering to a subject in need thereof a composition described herein, e.g., a cell genetically engineered using the fusion polypeptides and methods described herein, a population of cells or descendants thereof, or a pharmaceutical composition comprising the same. The cell, population of cells, or descendants thereof may comprise one or more modifications (e.g., genetic modifications) relative to a wildtype cell. In some embodiments, the cell, population of cells, or descendants thereof comprise a modification to a first gene relative to a wildtype cell of the same type. In some embodiments, the cell, population of cells, or descendants thereof comprise a modification to a second gene relative to a wildtype cell of the same type. In some embodiments, the cell, population of cells, or descendants thereof may comprise one or more modifications (e.g., genetic modifications) relative to a disease cell, such as a cell associated with a disease or disorder (e.g., cancer cell). Genes modified may correspond to any genetic locus targetable by the methods described herein, such as any of the exemplary genes or proteins described herein.

In some embodiments, the methods further involve administering to the subject a therapeutically effective amount of at least one agent that targets a product encoded by a wildtype copy of the modified gene. Without wishing to be bound by theory, by administering an agent that targets a product encoded by a wildtype copy of the modified gene in combination with a cell, population of cells, or descendants thereof comprising the modified gene, it is possible to target cells within a subject with the agent (e.g., disease cells, e.g., cancer cells) while not targeting or targeting to a lesser degree the cell, population of cells, or descendants thereof. For example, such a method may be used to selectively ablate or kill a target cell population in a subject while in combination replenishing the subject with new cells not vulnerable to the agent. As a further example, such a method may administer the agent as a part of the cell, population of cells, or descendants thereof (e.g., a CAR-T therapeutic), and would thus avoid or decrease cell fratricide. In some embodiments, administration of the at least one agent targeting the product encoded by the wildtype copy of the modified gene occurs simultaneously or in temporal proximity with administration of the cell, population or descendant thereof, or the pharmaceutical composition. In some embodiments, administration of the at least one agent targeting the product encoded by the wildtype copy of the modified gene occurs after administration of the cell, population or descendant thereof, or the pharmaceutical composition. In some embodiments, administration of the at least one agent targeting the product encoded by the wildtype copy of the modified gene occurs before administration of the cell, population or descendant thereof, or the pharmaceutical composition. In some embodiments, where the cell, population of cells, or descendants thereof comprises a modification to a first gene and a second gene relative to a wildtype cell of the same type, the method may comprise administering one or more (e.g., two agents) targeting the products of the first gene and the second gene (e.g., wildtype copies of the first gene and the second gene).

A subject in need thereof is, in some embodiments, a subject undergoing or about to undergo an immunotherapy targeting a product of the first gene and/or second gene. A subject in need thereof is, in some embodiments, a subject having or having been diagnosed with, a malignancy, such as caner (e.g., cancer associated with the presence of cancer stem cells, a hematopoietic malignancy, a cancer characterized by expression of a product of the first and/or second gene. In some embodiments, a subject having such a malignancy may be a candidate for administration of the agent, such as an immunotherapeutic, targeting a product of the first gene and/or second gene, but the risk of detrimental on-target, off-disease effects may outweigh the benefit, expected or observed, to the subject. In some such embodiments, administration of genetically engineered cells as described herein, results in an amelioration of the detrimental on-target, off-disease effects, as the genetically engineered cells provided herein are not targeted efficiently by the agent.

In some embodiments, the malignancy is a hematologic malignancy, or a cancer of the blood. In some embodiments, the malignancy is a lymphoid malignancy or a myeloid malignancy.

In some embodiments, the malignancy is an autoimmune disease or disorder. Examples of autoimmune disorders include, without limitation, rheumatoid arthritis, multiple sclerosis, leukemia, graft-versus host disease, lupus, and psoriasis.

In some embodiments, the malignancy is graft-versus host disease.

Also within the scope of the present disclosure are malignancies that are considered to be relapsed and/or refractory, such as relapsed or refractory hematological malignancies. A subject in need thereof is, in some embodiments, a subject undergoing or that will undergo an immune effector cell therapy targeting a product of the first gene and/or second gene, e.g., CAR-T cell therapy, wherein the immune effector cells express a CAR targeting the product, and wherein at least a subset of the immune effector cells also express the product on their cell surface. As used herein, the term “fratricide” refers to self-killing. For example, cells of a population of cells kill or induce killing of cells of the same population. In some embodiments, cells of the immune effector cell therapy kill or induce killing of other cells of the immune effector cell therapy.

In such embodiments, fratricide ablates a portion of or the entire population of immune effector cells before a desired clinical outcome, e.g., ablation of malignant cells expressing the product within the subject, can be achieved. In some such embodiments, using genetically engineered immune effector cells, as provided herein, e.g., immune effector cells that do not express the product or do not express a variant of the product recognized by the CAR, as the immune effector cells forming the basis of the immune effector cell therapy, will avoid such fratricide and the associated negative impact on therapy outcome. In such embodiments, genetically engineered immune effector cells, as provided herein, e.g., immune effector cells that do not express the product or do not express a variant of the product recognized by the CAR, may be further modified to also express the agent (e.g., a CAR targeting the product). In some embodiments, the immune effector cells may be lymphocytes, e.g., T-lymphocytes, such as, for example alpha/beta T lymphocytes, gamma/delta T-lymphocytes, or natural killer T cells. In some embodiments, the immune effect or cells may be natural killer (NK) cells.

In some embodiments, an effective number of genetically engineered cells as described herein, comprising modifications in their genome is administered to a subject in need thereof, e.g., a subject undergoing or that will undergo a therapy targeting a product of the first gene and/or second gene, wherein the therapy is associated or is at risk of being associated with a detrimental on-target, off-disease effect, e.g., in the form of cytotoxicity towards healthy cells in the subject that express the product. In some embodiments, an effective number of such genetically engineered cells may be administered to the subject in combination with the agent targeting a product encoded by a first gene or a second gene.

It is understood that when genetically modified cells and agents targeting a product encoded by a first gene or a second gene (e.g., an immunotherapeutic agent) are administered in combination, the cells and the agent may be administered at the same time or at different times, e.g., in temporal proximity.

For example, in some embodiments, administration in combination includes administration in the same course of treatment, e.g., in the course of treating a subject with an agent targeting a product (e.g., immunotherapy), the subject may be administered an effective number of genetically engineered cells, simultaneously, concurrently, or sequentially, e.g., before, during, or after the treatment with the agent, and/or in any order with respect to each other and the cells, population of cells, or descendants thereof. Furthermore, the cells and the agent may be admixed or in separate volumes or dosage forms.

In some embodiments, the agent that targets a product encoded by the first gene or a wildtype copy thereof is an immunotherapeutic agent. In some embodiments, the agent that targets a product encoded by the first gene or a wild-type copy thereof comprises an antigen binding fragment that binds the product encoded by the first gene or a wildtype copy thereof. In some embodiments, the agent that targets a product encoded by the first gene or a wildtype copy thereof comprises an antigen binding fragment that binds the product encoded by the second gene or a wildtype copy thereof.

In some embodiments, the agent is an immune cell that expresses a chimeric antigen receptor, which comprises an antigen-binding fragment (e.g., a single-chain antibody) capable of binding to a product produced by the first gene or a wild-type copy thereof. In some embodiments, the agent is an immune cell that expresses a chimeric antigen receptor, which comprises an antigen-binding fragment (e.g., a single-chain antibody) capable of binding to a product produced by the second gene or a wild-type copy thereof. The immune cell may be, e.g., a T cell (e.g., a CD4+ or CD8+ T cell) or an NK cell.

A Chimeric Antigen Receptor (CAR) can comprise a recombinant polypeptide comprising at least an extracellular antigen binding domain, a transmembrane domain, and a cytoplasmic signaling domain comprising a functional signaling domain, e.g., one derived from a stimulatory molecule. In one some embodiments, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule, such as 4-1BB (i.e., CD137), CD27, and/or CD28, or fragments of those molecules. The extracellular antigen binding domain of the CAR may comprise an antibody fragment that binds a product encoded by the first gene or a wildtype copy thereof, a product encoded by the second gene or a wildtype copy thereof, or both. The antibody fragment can comprise one or more CDRs, the variable regions (or portions thereof), the constant regions (or portions thereof), or combinations of any of the foregoing.

A chimeric antigen receptor (CAR) typically comprises an antigen-binding domain, e.g., comprising an antibody fragment, fused to a CAR framework, which may comprise a hinge region (e.g., from CD8 or CD28), a transmembrane domain (e.g., from CD8 or CD28), one or more costimulatory domains (e.g., CD28 or 4- IBB), and a signaling domain (e.g., CD3zeta). Exemplary sequences of CAR domains and components are provided, for example in PCT Publication No. WO 2019/178382, and in Table 1 below.

Table 1: Exemplary components of a chimeric receptor

In some embodiments, the number of genetically engineered cells provided herein, e.g., HSCs, HPCs, or immune effector cells (e.g., CAR-expressing cells) that are administered to a subject in need thereof, is within the range of 10 ⁶ -10 ⁿ . However, amounts below or above this exemplary range are also within the scope of the present disclosure. For example, in some embodiments, the number of genetically engineered cells provided herein, e.g., HSCs, HPCs, or immune effector cells (e.g., CAR-expressing cells) that are administered to a subject in need thereof is about 10 ⁶ , about 10 ⁷ , about 10 ⁸ , about 10 ⁹ , about IO ¹⁰ , or about 10 ¹¹ . In some embodiments, the number of genetically engineered cells provided herein, e.g., HSCs, HPCs, or immune effector cells (e.g., CAR-expressing cells) that are administered to a subject in need thereof, is within the range of 10 ⁶ -10 ⁹ , within the range of 10 ⁶ -10 ⁸ , within the range of 10 ⁷ -10 ⁹ , within the range of about 1O ⁷ -1O ¹⁰ , within the range of 1O ⁸ -1O ¹⁰ , or within the range of 10 ⁹ -10 ⁿ .

In some embodiments, the agent that targets a product encoded by the first gene or a wildtype copy thereof is an antibody-drug conjugate (ADC). The ADC may be a molecule comprising an antibody or antigen-binding fragment thereof conjugated to a toxin or drug molecule. Binding of the antibody or fragment thereof to the corresponding antigen allows for delivery of the toxin or drug molecule to a cell that presents the antigen on the cell surface (e.g., target cell), thereby resulting in death of the target cell. Toxins or drugs compatible for use in antibody-drug conjugates are known in the art and will be evident to one of ordinary skill in the art. See, e.g., Peters et al. Biosci. Rep. (2015) 35(4): e00225; Beck et al. Nature Reviews Drug Discovery (2017) 16:315-337; Marin-Acevedo et al. J. Hematol. Oncol. (2018)11: 8; Elgundi et al. Advanced Drug Delivery Reviews (2017) 122: 2-19.

In some embodiments, the antibody-drug conjugate may further comprise a linker (e.g., a peptide linker, such as a cleavable linker) attaching the antibody and drug molecule.

Examples of suitable toxins or drugs for antibody-drug conjugates include, without limitation, the toxins and drugs comprised in brentuximab vedotin, glembatumumab vedotin/CDX-011, depatuxizumab mafodotin/ ABT-414, PSMA ADC, polatuzumab vedotin/RG7596/DCDS4501A, denintuzumab mafodotin/SGN-CD19A, AGS-16C3F, CDX- 014, RG7841/DLYE5953A, RG7882/DMUC406A, RG7986/DCDS0780A, SGN-LIV1A, enfortumab vedotin/ASG-22ME, AG-15ME, AGS67E, telisotuzumab vedotin/ ABB V-399, ABBV-221, ABBV-085, GSK-2857916, tisotumab vedotin/HuMax-TF-ADC, HuMax-Axl- ADC, pinatuzumab vedotin/RG7593/DCDT2980S, lifastuzumab vedotin/RG7599/DNIB0600A, indusatumab vedotin/MLN-0264/TAK-264, vandortuzumab vedotin/RG7450/DSTP3086S, sofituzumab vedotin/RG7458/DMUC5754A, RG7600/DMGT4039A, RG7336/DEDN6526A, ME1547, PF-06263507/ADC 5T4, trastuzumab emtansine/T-DMl, mirvetuximab soravtansine/ IMGN853, coltuximab ravtansine/SAR3419, naratuximab emtansine/IMGN529, indatuximab ravtansine/BT-062, anetumab ravtansine/BAY 94-9343, SAR408701, SAR428926, AMG 224, PCA062, HKT288, LY3076226, SAR566658, lorvotuzumab mertansine/IMGN901, cantuzumab mertansine/SB-408075, cantuzumab ravtansine/IMGN242, laprituximab emtansine/IMGN289, IMGN388, bivatuzumab mertansine, AVE9633, BIIB015, MLN2704, AMG 172, AMG 595, LOP 628, vadastuximab talirine/SGN-CD33A, SGN-CD70A, SGN- CD19B, SGN-CD123A, SGN-CD352A, rovalpituzumab tesirine/SC16LD6.5, SC-002, SC- 003, ADCT-301/HuMax-TAC-PBD, ADCT-402, MEDI3726/ADC-401, IMGN779, IMGN632, gemtuzumab ozogamicin, inotuzumab ozogamicin/ CMC-544, PF-06647263, CMD-193, CMB-401, trastuzumab duocarmazine/SYD985, BMS-936561/MDX-1203, sacituzumab govitecan/IMMU-132, labetuzumab govitecan/IMMU-130, DS-8201a, U3- 1402, milatuzumab doxorubicin/IMMU-110/hLLl-DOX, BMS-986148, RC48- ADC/hertuzumab-vc-MMAE, PF-06647020, PF-06650808, PF-06664178/RN927C, lupartumab amadotin/ BAY1129980, aprutumab ixadotin/BAYl 187982, ARX788, AGS62P1, XMT-1522, AbGn-107, MEDI4276, DSTA4637S/RG7861. Anti-CD30 antibody drug conjugates are known in the art, for example, Bradley et al. Am. J. Health Syst. Pharm. (2013) 70(7): 589-97; Shen et al. mAbs (2019) 11(6): 1149-1161.

In some embodiments, binding of the antibody-drug conjugate to an epitope of the cell-surface protein (e.g., cell-surface lineage-specific cell-surface protein) induces internalization of the antibody-drug conjugate, and the drug (or toxin) may be released intracellularly. In some embodiments, binding of the antibody-drug conjugate to the epitope of a cell-surface lineage- specific protein induces internalization of the toxin or drug, which allows the toxin or drug to kill the cells expressing the lineage- specific protein (target cells). In some embodiments, binding of the antibody-drug conjugate to the epitope of a cell-surface lineage- specific protein induces internalization of the toxin or drug, which may regulate the activity of the cell expressing the lineage- specific protein (target cells). The type of toxin or drug used in the antibody-drug conjugates described herein is not limited to any specific type.

Aspects of the disclosure also provide kits, for example kits comprising reagents, e.g., for producing a genetically engineered cell. In some embodiments, the kit comprises any of the fusion polypeptides described herein and a gRNA comprising a targeting domain complementary to a target sequence in the genome of a cell. In some embodiments, the fusion polypeptide and the gRNA form a ribonucleoprotein (RNP) complex under conditions suitable to bind a target domain in the genome of a cell or plurality of cells. In some embodiments, the kit comprises any of the fusion polypeptides described herein and a second gRNA comprising a targeting domain complementary to a second target sequence in the genome of a cell. In some embodiments, the second gRNA and fusion polypeptide form a ribonucleoprotein (RNP) complex under conditions suitable to bind a second target domain in the genome of a cell or plurality of cells.

In some embodiments, the kit comprises instructions for a method of contacting a cell or plurality of cells with a gRNA and any of the fusion polypeptides described herein. In some embodiments, the instructions provide that the cell or plurality of cells is contacted with the fusion polypeptide prior to contacting the cell or plurality of cells with the gRNA. In some embodiments, the instructions provide that the cell or plurality of cells is contacted with the gRNA prior to contacting the cell or plurality of cells with the fusion polypeptide.

In some embodiments, the kit comprises a cell or plurality of cells. In some embodiments, the kit does not comprise a cell or plurality of cells (e.g., the cell or plurality of cells recited by the instructions is acquired by other means). SEQUENCES

Nucleic acid sequences of exemplary vector sequences are provided below.

Construct A - (SEQ ID NO:45) gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagag ataattggaattaat ttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttctt gggtagtttgcagtt ttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgat ttcttggctttatat atcttgtggaaaggacgaaacaccgggtcttcgagaagacctgttttagagctagaaata gcaagttaaaataag gctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcttttttgttttagagct agaaatagcaagtta aaataaggctagtccgtttttagcgcgtgcgccaattctgcagacaaatggctctagagg tacccgttacataac ttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaatag taacgccaataggga ctttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatc aagtgtatcatatgc caagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattgtgcccagt acatgaccttatggg actttcctacttggcagtacatctacgtattagtcatcgctattaccatggtcgaggtga gccccacgttctgct tcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaat tattttgtgcagcga tgggggcggggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggg gcggggcgaggcgga gaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggc ggcggcggcggcggc cctataaaaagcgaagcgcgcggcgggcgggagtcgctgcgcgctgccttcgccccgtgc cccgctccgccgccg cctcgcgccgcccgccccggctctgactgaccgcgttactcccacaggtgagcgggcggg acggcccttctcctc cgggctgtaattagctgagcaagaggtaagggtttaagggatggttggttggtggggtat taatgtttaattacc tggagcacctgcctgaaatcactttttttcaggttggaccggtgccaccatgcaactggt gaagagcgagctgga agagaagaaaagcgagctcagacataagctgaagtacgttccccacgaatacattgaact gatagaaatcgctag aaacagtacgcaagacagaatactggaaatgaaggtgatggagttcttcatgaaggttta cggctatcgtggcaa acacctcgggggctcccggaagcccgacggggctatctacaccgtgggcagtcccatcga ctatggcgtgatcgt ggacaccaaagcttatagcggcggatataatctccccatcggccaagccgatgagatgca gaggtatgtggagga gaaccaaacaagaaacaagcatatcaaccccaacgagtggtggaaggtttatcctagctc ggtgaccgagtttaa gttcctattcgtgtctggccacttcaagggcaactataaggcacagctcactagactgaa tcatatcacgaattg caacggcgccgtgttatccgtggaggagctactgatcggcggagagatgatcaaagccgg caccctgaccctgga agaggtgagaagaaagtttaacaatggcgaaataaatttcggcagcggaagtggaagcgg ctccatcactagaac caccaaccctagaaacgtggtgcccaagatctacatgagcgccggcagcatccccctgac cacccacatcaccaa ctcaattcagcccaccctgtggaccatcggcagcatcaacggcgtggcccccctggccaa gagcatcaagctggg catccccgtgaccggcagcgcctacaccgatcagaccaccgccatggtgagaaagaaggt gagcgtgttcatggg cagcggcagcgggagcggctcatcgcagctggttaagagcgagttagaagaaaaaaagag cgaactgcggcataa actgaagtatgtcccacacgagtacatcgaactgatcgagatcgcgagaaactctaccca agacagaattctgga gatgaaagtaatggaatttttcatgaaggtgtatggatatagagggaagcacctgggtgg cagcagaaaacccgc cggcgccatctacactgtggggagccccatagactatggtgtgatcgtggataccaaggc gtatagcggcggtta caatctgcccattgggcaagcggacgagatgcaaagatatgtggaagagaatcagacgag gaacaagcacattaa ccctaatgagtggtggaaggtctaccctagctccgttaccgagttcaagttcctgtttgt gagcgggcattttaa gggcaactacaaggcacagctgacccgcctgaaccacataacaaactgcaacggtgccgt gctgagcgtagaaga gttgctaatcggcggcgagatgatcaaggccggcacgctaaccctcgaagaggtgcgcag aaagttcaataacgg cgaaatcaatttcagcggcagcgagactcccgggacctcagagtccgccacacccgaaag tacacagttcgaggg ctttaccaacctgtatcaggtgagcaagacactgcggtttgagctgatcccacagggcaa gaccctgaagcacat ccaggagcagggcttcatcgaggaggacaaggcccgcaatgatcactacaaggagctgaa gcccatcatcgatcg gatctacaagacctatgccgaccagtgcctgcagctggtgcagctggattgggagaacct gagcgccgccatcga ctcctatagaaaggagaaaaccgaggagacaaggaacgccctgatcgaggagcaggccac atatcgcaatgccat ccacgactacttcatcggccggacagacaacctgaccgatgccatcaataagagacacgc cgagatctacaaggg cctgttcaaggccgagctgtttaatggcaaggtgctgaagcagctgggcaccgtgaccac aaccgagcacgagaa cgccctgctgcggagcttcgacaagtttacaacctacttctccggcttttatagaaacag gaagaacgtgttcag cgccgaggatatcagcacagccatcccacaccgcatcgtgcaggacaacttccccaagtt taaggagaattgtca catcttcacacgcctgatcaccgccgtgcccagcctgcgggagcactttgagaacgtgaa gaaggccatcggcat cttcgtgagcacctccatcgaggaggtgttttccttccctttttataaccagctgctgac acagacccagatcga cctgtataaccagctgctgggaggaatctctcgggaggcaggcaccgagaagatcaaggg cctgaacgaggtgct gaatctggccatccagaagaatgatgagacagcccacatcatcgcctccctgccacacag attcatccccctgtt taagcagatcctgtccgataggaacaccctgtctttcatcctggaggagtttaagagcga cgaggaagtgatcca gtccttctgcaagtacaagacactgctgagaaacgagaacgtgctggagacagccgaggc cctgtttaacgagct gaacagcatcgacctgacacacatcttcatcagccacaagaagctggagacaatcagcag cgccctgtgcgacca ctgggatacactgaggaatgccctgtatgagcggagaatctccgagctgacaggcaagat caccaagtctgccaa ggagaaggtgcagcgcagcctgaagcacgaggatatcaacctgcaggagatcatctctgc cgcaggcaaggagct gagcgaggccttcaagcagaaaaccagcgagatcctgtcccacgcacacgccgccctgga tcagccactgcctac aaccctgaagaagcaggaggagaaggagatcctgaagtctcagctggacagcctgctggg cctgtaccacctgct ggactggtttgccgtggatgagtccaacgaggtggaccccgagttctctgcccggctgac cggcatcaagctgga gatggagccttctctgagcttctacaacaaggccagaaattatgccaccaagaagcccta ctccgtggagaagtt caagctgaactttcagatgcctacactggccagaggctgggacgtgaatgtggagaagaa cagaggcgccatcct gtttgtgaagaacggcctgtactatctgggcatcatgccaaagcagaagggcaggtataa ggccctgagcttcga gcccacagagaaaaccagcgagggctttgataagatgtactatgactacttccctgatgc cgccaagatgatccc aaagtgcagcacccagctgaaggccgtgacagcccactttcagacccacacaacccccat cctgctgtccaacaa tttcatcgagcctctggagatcacaaaggagatctacgacctgaacaatcctgagaagga gccaaagaagtttca gacagcctacgccaagaaaaccggcgaccagaagggctacagagaggccctgtgcaagtg gatcgacttcacaag ggattttctgtccaagtataccaagacaacctctatcgatctgtctagcctgcggccatc ctctcagtataagga cctgggcgagtactatgccgagctgaatcccctgctgtaccacatcagcttccagagaat cgccgagaaggagat catggatgccgtggagacaggcaagctgtacctgttccagatctataacaaggactttgc caagggccaccacgg caagcctaatctgcacacactgtattggaccggcctgttttctccagagaacctggccaa gacaagcatcaagct gaatggccaggccgagctgttctaccgccctaagtccaggatgaagaggatggcacaccg gctgggagagaagat gctgaacaagaagctgaaggatcagaaaaccccaatccccgacaccctgtaccaggagct gtacgactatgtgaa tcacagactgtcccacgacctgtctgatgaggccagggccctgctgcccaacgtgatcac caaggaggtgtctca cgagatcatcaaggataggcgctttaccagcgacaagttctttttccacgtgcctatcac actgaactatcaggc cgccaattccccatctaagttcaaccagagggtgaatgcctacctgaaggagcaccccga gacacctatcatcgg catcgcccggggcgagagaaacctgatctatatcacagtgatcgactccaccggcaagat cctggagcagcggag cctgaacaccatccagcagtttgattaccagaagaagctggacaacagggagaaggagag ggtggcagcaaggca ggcctggtctgtggtgggcacaatcaaggatctgaagcagggctatctgagccaggtcat ccacgagatcgtgga cctgatgatccactaccaggccgtggtggtgctggagaacctgaatttcggctttaagag caagaggaccggcat cgccgagaaggccgtgtaccagcagttcgagaagatgctgatcgataagctgaattgcct ggtgctgaaggacta tccagcagagaaagtgggaggcgtgctgaacccataccagctgacagaccagttcacctc ctttgccaagatggg cacccagtctggcttcctgttttacgtgcctgccccatatacatctaagatcgatcccct gaccggcttcgtgga ccccttcgtgtggaaaaccatcaagaatcacgagagccgcaagcacttcctggagggctt cgactttctgcacta cgacgtgaaaaccggcgacttcatcctgcactttaagatgaacagaaatctgtccttcca gaggggcctgcccgg ctttatgcctgcatgggatatcgtgttcgagaagaacgagacacagtttgacgccaaggg cacccctttcatcgc cggcaagagaatcgtgccagtgatcgagaatcacagattcaccggcagataccgggacct gtatcctgccaacga gctgatcgccctgctggaggagaagggcatcgtgttcagggatggctccaacatcctgcc aaagctgctggagaa tgacgattctcacgccatcgacaccatggtggccctgatccgcagcgtgctgcagatgcg gaactccaatgccgc cacaggcgaggactatatcaacagccccgtgcgcgatctgaatggcgtgtgcttcgactc ccggtttcagaaccc agagtggcccatggacgccgatgccaatggcgcctaccacatcgccctgaagggccagct gctgctgaatcacct gaaggagagcaaggatctgaagctgcagaacggcatctccaatcaggactggctggccta catccaggagctgcg caacaaaaggccggcggccacgaaaaaggccggccaggcaaaaaagaaaaagggatccta cccatacgatgttcc agattacgcttatccctacgacgtgcctgattatgcatacccatatgatgtccccgacta tgccgagggcagagg aagtctgctaacatgcggtgacgtcgaggagaatcctggcccaatgaccgagtacaagcc cacggtgcgcctcgc cacccgcgacgacgtccccagggccgtacgcaccctcgccgccgcgttcgccgactaccc cgccacgcgccacac cgtcgatccggaccgccacatcgagcgggtcaccgagctgcaagaactcttcctcacgcg cgtcgggctcgacat cggcaaggtgtgggtcgcggacgacggcgccgcggtggcggtctggaccacgccggagag cgtcgaagcgggggc ggtgttcgccgagatcggcccgcgcatggccgagttgagcggttcccggctggccgcgca gcaacagatggaagg cctcctggcgccgcaccggcccaaggagcccgcgtggttcctggccaccgtcggagtctc gcccgaccaccaggg caagggtctgggcagcgccgtcgtgctccccggagtggaggcggccgagcgcgccggggt gcccgccttcctgga gacctccgcgccccgcaacctccccttctacgagcggctcggcttcaccgtcaccgccga cgtcgaggtgcccga aggaccgcgcacctggtgcatgacccgcaagcccggtgcctgaactagtcctgcaggcat gcaagcttgatatca agcttatcgataatcaacctctggattacaaaatttgtgaaagattgactggtattctta actatgttgctcctt ttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatgg ctttcattttctcct ccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaac gtggcgtggtgtgca ctgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctccttt ccgggactttcgctt tccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacag gggctcggctgttgg gcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcct gtgttgccacctgga ttctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttcctt cccgcggcctgctgc cggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctcccttt gggccgcctccccgc atcgataccgtcgacctcgagggaattaattcgagctcggtacctttaagaccgatgact tacaaggcagctgta gatcttagccactttttaaaagaaattaactgtgccttctagttgccagccatctgttgt ttgcccctcccccgt gccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaat tgcatcgcattgtct gagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattg ggaagagaatagcag gcatgctggggagcggccgcaggaacccctagtgatggagttggccactccctctctgcg cgctcgctcgctcac tgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgag cgagcgagcgcgcag ctgcctgcaggggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcaca ccgcatacgtcaaag caaccatagtacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgc agcgtgaccgctaca cttgccagcgccttagcgcccgctcctttcgctttcttcccttcctttctcgccacgttc gccggctttccccgt caagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgac cccaaaaaacttgat ttgggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacg ttggagtccacgttc tttaatagtggactcttgttccaaactggaacaacactcaactctatctcgggctattct tttgatttataaggg attttgccgatttcggtctattggttaaaaaatgagctgatttaacaaaaatttaacgcg aattttaacaaaata ttaacgtttacaattttatggtgcactctcagtacaatctgctctgatgccgcatagtta agccagccccgacac ccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacaga caagctgtgaccgtc tccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaag ggcctcgtgatacgc ctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcactttt cggggaaatgtgcgc ggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaa taaccctgataaatg cttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttatt cccttttttgcggca ttttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagat cagttgggtgcacga gtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaa gaacgttttccaatg atgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaa gagcaactcggtcgc cgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatctt acggatggcatgaca gtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttactt ctgacaacgatcgga ggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgat cgttgggaaccggag ctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaaca acgttgcgcaaacta ttaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcg gataaagttgcagga ccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggt gagcgtggaagccgc ggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacg acggggagtcaggca actatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattgg taactgtcagaccaa gtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctag gtgaagatccttttt gataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagacccc gtagaaaagatcaaa ggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaacca ccgctaccagcggtg gtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcaga gcgcagataccaaat actgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcct acatacctcgctctg ctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggac tcaagacgatagtta ccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggag cgaacgacctacacc gaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaag gcggacaggtatccg gtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctgg tatctttatagtcct gtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcgg agcctatggaaaaac gccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgt

Construct B - (SEQ ID NO:46) gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagag ataattggaattaat ttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttctt gggtagtttgcagtt ttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgat ttcttggctttatat atcttgtggaaaggacgaaacaccgggtcttcgagaagacctgttttagagctagaaata gcaagttaaaataag gctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcttttttgttttagagct agaaatagcaagtta aaataaggctagtccgtttttagcgcgtgcgccaattctgcagacaaatggctctagagg tacccgttacataac ttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaatag taacgccaataggga ctttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatc aagtgtatcatatgc caagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattgtgcccagt acatgaccttatggg actttcctacttggcagtacatctacgtattagtcatcgctattaccatggtcgaggtga gccccacgttctgct tcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaat tattttgtgcagcga tgggggcggggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggg gcggggcgaggcgga gaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggc ggcggcggcggcggc cctataaaaagcgaagcgcgcggcgggcgggagtcgctgcgcgctgccttcgccccgtgc cccgctccgccgccg cctcgcgccgcccgccccggctctgactgaccgcgttactcccacaggtgagcgggcggg acggcccttctcctc cgggctgtaattagctgagcaagaggtaagggtttaagggatggttggttggtggggtat taatgtttaattacc tggagcacctgcctgaaatcactttttttcaggttggaccggtgccaccatgggcagctc agagactggcccagt ggctgtggaccccacattgagacggcggatcgagccccatgagtttgaggtattcttcga tccgagagagctccg caaggagacctgcctgctttacgaaattaattgggggggccggcactccatttggcgaca tacatcacagaacac taacaagcacgtcgaagtcaacttcatcgagaagttcacgacagaaagatatttctgtcc gaacacaaggtgcag cattacctggtttctcagctggagcccatgcggcgaatgtagtagggccatcactgaatt cctgtcaaggtatcc ccacgtcactctgtttatttacatcgcaaggctgtaccaccacgctgacccccgcaatcg acaaggcctgcggga tttgatctcttcaggtgtgactatccaaattatgactgagcaggagtcaggatactgctg gagaaactttgtgaa ttatagcccgagtaatgaagcccactggcctaggtatccccatctgtgggtacgactgta cgttcttgaactgta ctgcatcatactgggcctgcctccttgtctcaacattctgagaaggaagcagccacagct gacattctttaccat cgctcttcagtcttgtcattaccagcgactgcccccacacattctctgggccaccgggtt gaaatctggtggttc ttctggtggttctagcggcagcgagactcccgggacctcagagtccgccacacccgaaag ttccggagggagtag cggcgggtctacacagttcgagggctttaccaacctgtatcaggtgagcaagacactgcg gtttgagctgatccc acagggcaagaccctgaagcacatccaggagcagggcttcatcgaggaggacaaggcccg caatgatcactacaa ggagctgaagcccatcatcgatcggatctacaagacctatgccgaccagtgcctgcagct ggtgcagctggattg ggagaacctgagcgccgccatcgactcctatagaaaggagaaaaccgaggagacaaggaa cgccctgatcgagga gcaggccacatatcgcaatgccatccacgactacttcatcggccggacagacaacctgac cgatgccatcaataa gagacacgccgagatctacaagggcctgttcaaggccgagctgtttaatggcaaggtgct gaagcagctgggcac cgtgaccacaaccgagcacgagaacgccctgctgcggagcttcgacaagtttacaaccta cttctccggctttta tagaaacaggaagaacgtgttcagcgccgaggatatcagcacagccatcccacaccgcat cgtgcaggacaactt ccccaagtttaaggagaattgtcacatcttcacacgcctgatcaccgccgtgcccagcct gcgggagcactttga gaacgtgaagaaggccatcggcatcttcgtgagcacctccatcgaggaggtgttttcctt ccctttttataacca gctgctgacacagacccagatcgacctgtataaccagctgctgggaggaatctctcggga ggcaggcaccgagaa gatcaagggcctgaacgaggtgctgaatctggccatccagaagaatgatgagacagccca catcatcgcctccct gccacacagattcatccccctgtttaagcagatcctgtccgataggaacaccctgtcttt catcctggaggagtt taagagcgacgaggaagtgatccagtccttctgcaagtacaagacactgctgagaaacga gaacgtgctggagac agccgaggccctgtttaacgagctgaacagcatcgacctgacacacatcttcatcagcca caagaagctggagac aatcagcagcgccctgtgcgaccactgggatacactgaggaatgccctgtatgagcggag aatctccgagctgac aggcaagatcaccaagtctgccaaggagaaggtgcagcgcagcctgaagcacgaggatat caacctgcaggagat catctctgccgcaggcaaggagctgagcgaggccttcaagcagaaaaccagcgagatcct gtcccacgcacacgc cgccctggatcagccactgcctacaaccctgaagaagcaggaggagaaggagatcctgaa gtctcagctggacag cctgctgggcctgtaccacctgctggactggtttgccgtggatgagtccaacgaggtgga ccccgagttctctgc ccggctgaccggcatcaagctggagatggagccttctctgagcttctacaacaaggccag aaattatgccaccaa gaagccctactccgtggagaagttcaagctgaactttcagatgcctacactggccagagg ctgggacgtgaatgt ggagaagaacagaggcgccatcctgtttgtgaagaacggcctgtactatctgggcatcat gccaaagcagaaggg caggtataaggccctgagcttcgagcccacagagaaaaccagcgagggctttgataagat gtactatgactactt ccctgatgccgccaagatgatcccaaagtgcagcacccagctgaaggccgtgacagccca ctttcagacccacac aacccccatcctgctgtccaacaatttcatcgagcctctggagatcacaaaggagatcta cgacctgaacaatcc tgagaaggagccaaagaagtttcagacagcctacgccaagaaaaccggcgaccagaaggg ctacagagaggccct gtgcaagtggatcgacttcacaagggattttctgtccaagtataccaagacaacctctat cgatctgtctagcct gcggccatcctctcagtataaggacctgggcgagtactatgccgagctgaatcccctgct gtaccacatcagctt ccagagaatcgccgagaaggagatcatggatgccgtggagacaggcaagctgtacctgtt ccagatctataacaa ggactttgccaagggccaccacggcaagcctaatctgcacacactgtattggaccggcct gttttctccagagaa cctggccaagacaagcatcaagctgaatggccaggccgagctgttctaccgccctaagtc caggatgaagaggat ggcacaccggctgggagagaagatgctgaacaagaagctgaaggatcagaaaaccccaat ccccgacaccctgta ccaggagctgtacgactatgtgaatcacagactgtcccacgacctgtctgatgaggccag ggccctgctgcccaa cgtgatcaccaaggaggtgtctcacgagatcatcaaggataggcgctttaccagcgacaa gttctttttccacgt gcctatcacactgaactatcaggccgccaattccccatctaagttcaaccagagggtgaa tgcctacctgaagga gcaccccgagacacctatcatcggcatcgcccggggcgagagaaacctgatctatatcac agtgatcgactccac cggcaagatcctggagcagcggagcctgaacaccatccagcagtttgattaccagaagaa gctggacaacaggga gaaggagagggtggcagcaaggcaggcctggtctgtggtgggcacaatcaaggatctgaa gcagggctatctgag ccaggtcatccacgagatcgtggacctgatgatccactaccaggccgtggtggtgctgga gaacctgaatttcgg ctttaagagcaagaggaccggcatcgccgagaaggccgtgtaccagcagttcgagaagat gctgatcgataagct gaattgcctggtgctgaaggactatccagcagagaaagtgggaggcgtgctgaacccata ccagctgacagacca gttcacctcctttgccaagatgggcacccagtctggcttcctgttttacgtgcctgcccc atatacatctaagat cgatcccctgaccggcttcgtggaccccttcgtgtggaaaaccatcaagaatcacgagag ccgcaagcacttcct ggagggcttcgactttctgcactacgacgtgaaaaccggcgacttcatcctgcactttaa gatgaacagaaatct gtccttccagaggggcctgcccggctttatgcctgcatgggatatcgtgttcgagaagaa cgagacacagtttga cgccaagggcacccctttcatcgccggcaagagaatcgtgccagtgatcgagaatcacag attcaccggcagata ccgggacctgtatcctgccaacgagctgatcgccctgctggaggagaagggcatcgtgtt cagggatggctccaa catcctgccaaagctgctggagaatgacgattctcacgccatcgacaccatggtggccct gatccgcagcgtgct gcagatgcggaactccaatgccgccacaggcgaggactatatcaacagccccgtgcgcga tctgaatggcgtgtg cttcgactcccggtttcagaacccagagtggcccatggacgccgatgccaatggcgccta ccacatcgccctgaa gggccagctgctgctgaatcacctgaaggagagcaaggatctgaagctgcagaacggcat ctccaatcaggactg gctggcctacatccaggagctgcgcaacagcggcagcgagactcccgggacctcagagtc cgccacacccgaaag tcaactggtgaagagcgagctggaagagaagaaaagcgagctcagacataagctgaagta cgttccccacgaata cattgaactgatagaaatcgctagaaacagtacgcaagacagaatactggaaatgaaggt gatggagttcttcat gaaggtttacggctatcgtggcaaacacctcgggggctcccggaagcccgccggggctat ctacaccgtgggcag tcccatcgactatggcgtgatcgtggacaccaaagcttatagcggcggatataatctccc catcggccaagccga tgagatgcagaggtatgtggaggagaaccaaacaagaaacaagcatatcaaccccaacga gtggtggaaggttta tcctagctcggtgaccgagtttaagttcctattcgtgtctggccacttcaagggcaacta taaggcacagctcac tagactgaatcatatcacgaattgcaacggcgccgtgttatccgtggaggagctactgat cggcggagagatgat caaagccggcaccctgaccctggaagaggtgagaagaaagtttaacaatggcgaaataaa tttcggcagcggaag tggaagcggctccatcactagaaccaccaaccctagaaacgtggtgcccaagatctacat gagcgccggcagcat ccccctgaccacccacatcaccaactcaattcagcccaccctgtggaccatcggcagcat caacggcgtggcccc cctggccaagagcatcaagctgggcatccccgtgaccggcagcgcctacaccgatcagac caccgccatggtgag aaagaaggtgagcgtgttcatgggcagcggcagcgggagcggctcatcgcagctggttaa gagcgagttagaaga aaaaaagagcgaactgcggcataaactgaagtatgtcccacacgagtacatcgaactgat cgagatcgcgagaaa ctctacccaagacagaattctggagatgaaagtaatggaatttttcatgaaggtgtatgg atatagagggaagca cctgggtggcagcagaaaacccgacggcgccatctacactgtggggagccccatagacta tggtgtgatcgtgga taccaaggcgtatagcggcggttacaatctgcccattgggcaagcggacgagatgcaaag atatgtggaagagaa tcagacgaggaacaagcacattaaccctaatgagtggtggaaggtctaccctagctccgt taccgagttcaagtt cctgtttgtgagcgggcattttaagggcaactacaaggcacagctgacccgcctgaacca cataacaaactgcaa cggtgccgtgctgagcgtagaagagttgctaatcggcggcgagatgatcaaggccggcac gctaaccctcgaaga ggtgcgcagaaagttcaataacggcgaaatcaatttcaaaaggccggcggccacgaaaaa ggccggccaggcaaa aaagaaaaagggatcctacccatacgatgttccagattacgcttatccctacgacgtgcc tgattatgcataccc atatgatgtccccgactatgccgagggcagaggaagtctgctaacatgcggtgacgtcga ggagaatcctggccc aatgaccgagtacaagcccacggtgcgcctcgccacccgcgacgacgtccccagggccgt acgcaccctcgccgc cgcgttcgccgactaccccgccacgcgccacaccgtcgatccggaccgccacatcgagcg ggtcaccgagctgca agaactcttcctcacgcgcgtcgggctcgacatcggcaaggtgtgggtcgcggacgacgg cgccgcggtggcggt ct ggaccacgccggagagcgtcgaagcgggggcggtgttcgccgagatcggcccgcgcatgg ccgagtt gagegg ttcccggctggccgcgcagcaacagatggaaggcctcctggcgccgcaccggcccaagga gcccgcgtggttcct ggccaccgtcggagtctcgcccgaccaccagggcaagggtctgggcagcgccgtcgtgct ccccggagtggaggc ggccgagcgcgccggggtgcccgccttcctggagacctccgcgccccgcaacctcccctt ctacgagcggctcgg cttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcgcacctggtgcatgacccg caagcccggtgcctg aactagtcctgcaggcatgcaagcttgatatcaagcttatcgataatcaacctctggatt acaaaatttgtgaaa gattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaa tgcctttgtatcatg ctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctc tttatgaggagttgt ggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactg gttggggcattgcca ccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaac tcatcgccgcctgcc ttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcgg ggaaatcatcgtcct ttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacg tcccttcggccctca atccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttc gccttcgccctcaga cgagtcggatctccctttgggccgcctccccgcatcgataccgtcgacctcgagggaatt aattcgagctcggta cctttaagaccgatgacttacaaggcagctgtagatcttagccactttttaaaagaaatt aactgtgccttctag ttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccac tcccactgtcctttc ctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctgggggg tggggtggggcagga cagcaagggggaggattgggaagagaatagcaggcatgctggggagcggccgcaggaacc cctagtgatggagtt ggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccg acgcccgggctttgc ccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcaggggcgcctgatgcggta ttttctccttacgca tctgtgcggtatttcacaccgcatacgtcaaagcaaccatagtacgcgccctgtagcggc gcattaagcgcggcg ggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccttagcgcccgctcct ttcgctttcttccct tcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctcccttta gggttccgatttagt gctttacggcacctcgaccccaaaaaacttgatttgggtgatggttcacgtagtgggcca tcgccctgatagacg gtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaact ggaacaacactcaac tctatctcgggctattcttttgatttataagggattttgccgatttcggtctattggtta aaaaatgagctgatt taacaaaaatttaacgcgaattttaacaaaatattaacgtttacaattttatggtgcact ctcagtacaatctgc tctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctga cgggcttgtctgctc ccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttt tcaccgtcatcaccg aaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgata ataatggtttcttag acgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaa atacattcaaatatg tatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagt atgagtattcaacat ttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcaccca gaaacgctggtgaaa gtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaac agcggtaagatcctt gagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgt ggcgcggtattatcc cgt at tgacgccgggcaagagcaactcggtcgccgcatacact at t ct cagaatgacttggtt gagtact caeca gtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccata accatgagtgataac actgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttg cacaacatgggggat catgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgag cgtgacaccacgatg cctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagct tcccggcaacaatta atagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggct ggctggtttattgct gataaatctggagccggtgagcgtggaagccgcggtatcattgcagcactggggccagat ggtaagccctcccgt atcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatc gctgagataggtgcc tcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgat ttaaaacttcatttt taatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaa cgtgagttttcgttc cactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctg cgcgtaatctgctgc ttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctacca actctttttccgaag gtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagtta ggccaccacttcaag aactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgcc agtggcgataagtcg tgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctga acggggggttcgtgc acacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagcta tgagaaagcgccacg cttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagag cgcacgagggagctt ccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgag cgtcgatttttgtga tgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttc ctggccttttgctgg ccttttgctcacatgt

Construct C (SEQ. ID NO:47) gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagag ataattggaattaat ttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttctt gggtagtttgcagtt ttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgat ttcttggctttatat atcttgtggaaaggacgaaacaccgggtcttcgagaagacctgttttagagctagaaata gcaagttaaaataag gctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcttttttgttttagagct agaaatagcaagtta aaataaggctagtccgtttttagcgcgtgcgccaattctgcagacaaatggctctagagg tacccgttacataac ttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaatag taacgccaataggga ctttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatc aagtgtatcatatgc caagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattgtgcccagt acatgaccttatggg act t t cct act t ggcagt acat ct aegt at tagteat eget at taccatggtcgaggtgagccccacgtt ct get tcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaat tattttgtgcagcga tgggggcggggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggg gcggggcgaggcgga gaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggc ggcggcggcggcggc cctataaaaagcgaagcgcgcggcgggcgggagtcgctgcgcgctgccttcgccccgtgc cccgctccgccgccg cctcgcgccgcccgccccggctctgactgaccgcgttactcccacaggtgagcgggcggg acggcccttctcctc cgggctgtaattagctgagcaagaggtaagggtttaagggatggttggttggtggggtat taatgtttaattacc tggagcacctgcctgaaatcactttttttcaggttggaccggtgccaccatgggcagctc agagactggcccagt ggctgtggaccccacattgagacggcggatcgagccccatgagtttgaggtattcttcga tccgagagagctccg caaggagacctgcct get ttacgaaattaattgggggggccggcactccatttggcgacat acat cacagaacac taacaagcacgtcgaagtcaacttcatcgagaagttcacgacagaaagatatttctgtcc gaacacaaggtgcag cattacctggtttctcagctggagcccatgcggcgaatgtagtagggccatcactgaatt cctgtcaaggtatcc ccacgtcactctgtttatttacatcgcaaggctgtaccaccacgctgacccccgcaatcg acaaggcctgcggga tttgatctcttcaggtgtgactatccaaattatgactgagcaggagtcaggatactgctg gagaaactttgtgaa ttatagcccgagtaatgaagcccactggcctaggtatccccatctgtgggtacgactgta cgttcttgaactgta ctgcatcatactgggcctgcctccttgtctcaacattctgagaaggaagcagccacagct gacattctttaccat cgctcttcagtcttgtcattaccagcgactgcccccacacattctctgggccaccgggtt gaaatctggtggttc ttctggtggttctagcggcagcgagactcccgggacctcagagtccgccacacccgaaag ttccggagggagtag cggcgggtctaataacggaactaataacttccaaaacttcatcgggatcagttccttgca gaaaactctccggaa tgctctcatcccaactgagactactcagcagttcattgttaagaatggaatcataaaaga ggacgagcttagggg ggaaaataggcaaatcctcaaggatatcatggatgactattataggggctttatatccga gacactgagcagcat tgatgatatagactggacctctcttttcgaaaagatggaaatacaacttaaaaatggaga taacaaggacaccct gataaaggaacagaccgaatataggaaggcaattcataaaaagtttgctaacgatgatag gtttaaaaacatgtt ctcagcaaaactcatttcagatatactgcccgaattcgttatccacaacaacaactactc cgctagcgaaaaaga ggaaaagacccaagtcataaagctgttctctcgattcgcgacgagttttaaagattattt ccgaaatcgcgcaaa ctgtttctcagctgatgatatcagcagctcatcctgtcatcggatcgttaacgataatgc tgaaatcttcttctc caatgcacttgtttataggcgcattgttaaatctctctcaaacgatgatatcaataagat ttccggcgatatgaa ggacagtcttaaggagatgagcctcgaagagatatactcatacgagaaatatggcgaatt tatcacccaggaagg gatttccttctataatgacatttgcggcaaagtcaattccttcatgaacctgtattgcca aaaaaataaagaaaa caagaacctctataagctgcaaaagttgcataagcaaatactttgtatcgcggatacaag ctatgaagttcccta caagttcgagagtgatgaggaggtgtatcaatctgtcaatggtttccttgataatatttc ttctaagcatattgt tgaacgactccgaaagataggagacaactataatggatacaatttggataaaatctacat cgtgtctaaatttta cgagagtgtgtcacaaaaaacatatagagactgggagacaattaataccgccctggagat acattacaacaatat acttcccgggaacgggaagtctaaggcagacaaggtgaagaaagccgtgaagaacgactt gcaaaagtcaattac cgaaatcaatgagcttgtttcaaactataaactttgttcagatgacaatattaaagccga aacctatattcatga aatctctcatattctgaataactttgaggcgcaagaactgaaatataacccagaaataca cctcgttgagtccga actgaaagcaagcgaactgaaaaatgttttggacgtgataatgaacgcttttcattggtg ctcagtctttatgac agaggagcttgttgacaaggataacaatttctatgcggaactggaagagatttacgacga aatctatccggtcat atccctgtataacctggttcgcaactatgtcacgcaaaaaccatacagcacgaagaagat taaactgaactttgg tattccgacgctggcccgcggatggtcaaaatctgttgaatactcacgaaatgccataat cctgatgcgagataa cctctactaccttggaatctttaatgctaaaaataaacccgataaaaaaattatcgaagg gaacacgagtgaaaa caaaggtgattataaaaaaatgatatataatctgcttccaggaccaaataagatgatacc caaagttttcctttc ttcaaagaccggcgtcgagacatataaaccatccgcgtacatacttgaaggctacaaaca aaataaacatatcaa atcatctaaggattttgacattacgttctgtcatgatttgattgactatttcaaaaattg catagccattcatcc agagtggaaaaactttgggtttgacttctctgataccagtacatatgaagacataagtgg attttaccgagaagt agagctccaaggttataaaatagactggacctatatatctgaaaaggatatagacctttt gcaagagaagggaca gctttatcttttccaaatctacaacaaagacttcagtaagaaaagtaccgggaatgacaa tcttcataccatgta tctgaagaacctgttctccgaagaaaatctgaaggacatagtcctgaagcttaatggcga agcggaaattttttt ccgaaagagctctattaagaaccccataatacataagaagggaagcattctcgttaatcg aacgtatgaggccga agagaaagatcaatttgggaatatccaaatcgttcgaaagaacataccagaaaatattta ccaagaattgtacaa atattttaacgataaaagcgacaaagaactgtctgatgaagctgctaagctgaaaaacgt cgtcggccatcatga ggccgcgacgaatatagtcaaggattaccgatatacatacgataagtatttcctgcatat gcccatcactatcaa ctttaaggcaaataagactggattcattaatgacagaatactgcaatacatagctaaaga aaaagatttgcatgt tattggcattgccaggggtgagcgcaatcttatctatgtaagcgtcattgatacttgcgg gaatatcgtagagca gaagtcatttaatattgtaaatgggtacgattaccaaatcaagttgaagcagcaagaggg agcacgacagattgc ccgcaaggagtggaaagagatcggaaagataaaggagatcaaggaggggtatttgtccct tgttatacacgaaat ttccaagatggtaatcaagtacaacgctataattgctatggaggatctctcctatggatt taaaaagggaagatt taaagtcgagcggcaggtatatcagaaatttgaaacaatgcttattaataaacttaatta tctcgttttcaaaga cattagtatcaccgaaaacggtgggctgttgaagggctatcaacttacgtacataccaga taagcttaagaatgt gggtcaccaatgcggatgcatattctacgtgcccgcagcttatacaagcaaaatcgaccc aacaacgggtttcgt aaacatatttaagttcaaggatctcaccgtggatgccaagcgagagttcataaaaaaatt tgactcaatcagata tgactcagaaaagaatcttttttgttttaccttcgactacaataatttcattacacaaaa tacggttatgagcaa gtcatcctggtccgtatatacgtatggagtgcgcataaagcggagattcgttaacgggcg attttctaatgagtc cgatacaatcgatataacaaaggatatggaaaaaactctggaaatgactgatataaattg gagggacggtcatga cctcaggcaagacattatcgattatgagatcgtgcaacatatttttgagatctttcggtt gactgtccaaatgag gaactctctgtctgaattggaagatagggactacgatcgcctgataagccccgtgttgaa cgagaataacatatt ctacgattccgcgaaagccggggatgcgctccctaaggacgccgatgcaaatggggccta ttgtattgctttgaa agggctgtacgaaatcaaacagatcaccgaaaactggaaagaagacgggaagtttagtcg ggataaactgaagat atccaacaaggactggtttgactttatccaaaataagcgatatttgagcggcagcgagac tcccgggacctcaga gtccgccacacccgaaagtcaactggtgaagagcgagctggaagagaagaaaagcgagct cagacataagctgaa gtacgttccccacgaatacattgaactgatagaaatcgctagaaacagtacgcaagacag aatactggaaatgaa ggtgatggagttcttcatgaaggtttacggctatcgtggcaaacacctcgggggctcccg gaagcccgccggggc tatctacaccgtgggcagtcccatcgactatggcgtgatcgtggacaccaaagcttatag cggcggatataatct ccccatcggccaagccgatgagatgcagaggtatgtggaggagaaccaaacaagaaacaa gcatatcaaccccaa cgagtggtggaaggtttatcctagctcggtgaccgagtttaagttcctattcgtgtctgg ccacttcaagggcaa ctataaggcacagctcactagactgaatcatatcacgaattgcaacggcgccgtgttatc cgtggaggagctact gatcggcggagagatgatcaaagccggcaccctgaccctggaagaggtgagaagaaagtt taacaatggcgaaat aaatttcggcagcggaagtggaagcggctccatcactagaaccaccaaccctagaaacgt ggtgcccaagatcta catgagcgccggcagcatccccctgaccacccacatcaccaactcaattcagcccaccct gtggaccatcggcag catcaacggcgtggcccccctggccaagagcatcaagctgggcatccccgtgaccggcag cgcctacaccgatca gaccaccgccatggtgagaaagaaggtgagcgtgttcatgggcagcggcagcgggagcgg ctcatcgcagctggt taagagcgagttagaagaaaaaaagagcgaactgcggcataaactgaagtatgtcccaca cgagtacatcgaact gatcgagatcgcgagaaactctacccaagacagaattctggagatgaaagtaatggaatt tttcatgaaggtgta tggatatagagggaagcacctgggtggcagcagaaaacccgacggcgccatctacactgt ggggagccccataga ctatggtgtgatcgtggataccaaggcgtatagcggcggttacaatctgcccattgggca agcggacgagatgca aagatatgtggaagagaatcagacgaggaacaagcacattaaccctaatgagtggtggaa ggtctaccctagctc cgttaccgagttcaagttcctgtttgtgagcgggcattttaagggcaactacaaggcaca gctgacccgcctgaa ccacataacaaactgcaacggtgccgtgctgagcgtagaagagttgctaatcggcggcga gatgatcaaggccgg cacgctaaccctcgaagaggtgcgcagaaagttcaataacggcgaaatcaatttcaaaag gccggcggccacgaa aaaggccggccaggcaaaaaagaaaaagggatcctacccatacgatgttccagattacgc ttatccctacgacgt gcctgattatgcatacccatatgatgtccccgactatgccgagggcagaggaagtctgct aacatgcggtgacgt cgaggagaatcctggcccaatgaccgagtacaagcccacggtgcgcctcgccacccgcga cgacgtccccagggc cgtacgcaccctcgccgccgcgttcgccgactaccccgccacgcgccacaccgtcgatcc ggaccgccacatcga gcgggtcaccgagctgcaagaactcttcctcacgcgcgtcgggctcgacatcggcaaggt gtgggtcgcggacga cggcgccgcggtggcggtctggaccacgccggagagcgtcgaagcgggggcggtgttcgc cgagatcggcccgcg catggccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcctcctggc gccgcaccggcccaa ggagcccgcgtggttcctggccaccgtcggagtctcgcccgaccaccagggcaagggtct gggcagcgccgtcgt gctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgc gccccgcaacctccc cttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcg cacctggtgcatgac ccgcaagcccggtgcctgaactagtcctgcaggcatgcaagcttgatatcaagcttatcg ataatcaacctctgg attacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctat gtggatacgctgctt taatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtata aatcctggttgctgt ctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttg ctgacgcaaccccca ctggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctcc ctattgccacggcgg aactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgaca attccgtggtgttgt cggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcg ggacgtccttctgct acgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgc ggcctcttccgcgtc ttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcatcgatacc gtcgacctcgaggga attaattcgagctcggtacctttaagaccgatgacttacaaggcagctgtagatcttagc cactttttaaaagaa attaactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttcctt gaccctggaaggtgc cactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtg tcattctattctggg gggtggggtggggcaggacagcaagggggaggattgggaagagaatagcaggcatgctgg ggagcggccgcagga acccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgg gcgaccaaaggtcgc ccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgca ggggcgcctgatgcg gtattttctccttacgcatctgtgcggtatttcacaccgcatacgtcaaagcaaccatag tacgcgccctgtagc ggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagc gccttagcgcccgct cctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctcta aatcgggggctccct ttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgatttgggtgat ggttcacgtagtggg ccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagt ggactcttgttccaa actggaacaacactcaactctatctcgggctattcttttgatttataagggattttgccg atttcggtctattgg ttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgttt acaattttatggtgc actctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaaca cccgctgacgcgccc tgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagc tgcatgtgtcagagg ttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctattttta taggttaatgtcatg ataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccct atttgtttatttttc taaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataa tattgaaaaaggaag agtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgcctt cctgtttttgctcac ccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttac atcgaactggatctc aacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcact tttaaagttctgcta tgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacac tattctcagaatgac ttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaa ttatgcagtgctgcc ataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaag gagctaaccgctttt ttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaa gccataccaaacgac gagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggc gaactacttactcta gcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctg cgctcggcccttccg gctggctggtttattgctgataaatctggagccggtgagcgtggaagccgcggtatcatt gcagcactggggcca gatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggat gaacgaaatagacag atcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactca tatatactttagatt gatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctc atgaccaaaatccct taacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttct tgagatccttttttt ctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttg ccggatcaagagcta ccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttctt ctagtgtagccgtag ttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctg ttaccagtggctgct gccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataag gcgcagcggtcgggc tgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgaga tacctacagcgtgag ctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggc agggtcggaacagga gagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggttt cgccacctctgactt gagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaac gcggcctttttacgg ttcctggccttttgctggccttttgctcacatgt

Construct D .(SEQ ID NO:48) gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagag ataattggaattaat ttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttctt gggtagtttgcagtt ttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgat ttcttggctttatat atcttgtggaaaggacgaaacaccgggtcttcgagaagacctgttttagagctagaaata gcaagttaaaataag gctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcttttttgttttagagct agaaatagcaagtta aaataaggctagtccgtttttagcgcgtgcgccaattctgcagacaaatggctctagagg tacccgttacataac ttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaatag taacgccaataggga ctttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatc aagtgtatcatatgc caagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattgtgcccagt acatgaccttatggg actttcctacttggcagtacatctacgtattagtcatcgctattaccatggtcgaggtga gccccacgttctgct tcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaat tattttgtgcagcga tgggggcggggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggg gcggggcgaggcgga gaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggc ggcggcggcggcggc cctataaaaagcgaagcgcgcggcgggcgggagtcgctgcgcgctgccttcgccccgtgc cccgctccgccgccg cctcgcgccgcccgccccggctctgactgaccgcgttactcccacaggtgagcgggcggg acggcccttctcctc cgggctgtaattagctgagcaagaggtaagggtttaagggatggttggttggtggggtat taatgtttaattacc tggagcacctgcctgaaatcactttttttcaggttggaccggtgccaccatgaataacgg aactaataacttcca aaacttcatcgggatcagttccttgcagaaaactctccggaatgctctcatcccaactga gactactcagcagtt cattgttaagaatggaatcataaaagaggacgagcttaggggggaaaataggcaaatcct caaggatatcatgga tgactattataggggctttatatccgagacactgagcagcattgatgatatagactggac ctctcttttcgaaaa gatggaaatacaacttaaaaatggagataacaaggacaccctgataaaggaacagaccga atataggaaggcaat tcataaaaagtttgctaacgatgataggtttaaaaacatgttctcagcaaaactcatttc agatatactgcccga attcgttatccacaacaacaactactccgctagcgaaaaagaggaaaagacccaagtcat aaagctgttctctcg attcgcgacgagttttaaagattatttccgaaatcgcgcaaactgtttctcagctgatga tatcagcagctcatc ctgtcatcggatcgttaacgataatgctgaaatcttcttctccaatgcacttgtttatag gcgcattgttaaatc tctctcaaacgatgatatcaataagatttccggcgatatgaaggacagtcttaaggagat gagcctcgaagagat atactcatacgagaaatatggcgaatttatcacccaggaagggatttccttctataatga catttgcggcaaagt caattccttcatgaacctgtattgccaaaaaaataaagaaaacaagaacctctataagct gcaaaagttgcataa gcaaatactttgtatcgcggatacaagctatgaagttccctacaagttcgagagtgatga ggaggtgtatcaatc tgtcaatggtttccttgataatatttcttctaagcatattgttgaacgactccgaaagat aggagacaactataa tggatacaatttggataaaatctacatcgtgtctaaattttacgagagtgtgtcacaaaa aacatatagagactg ggagacaattaataccgccctggagatacattacaacaatatacttcccgggaacgggaa gtctaaggcagacaa ggtgaagaaagccgtgaagaacgacttgcaaaagtcaattaccgaaatcaatgagcttgt ttcaaactataaact ttgttcagatgacaatattaaagccgaaacctatattcatgaaatctctcatattctgaa taactttgaggcgca agaactgaaatataacccagaaatacacctcgttgagtccgaactgaaagcaagcgaact gaaaaatgttttgga cgtgataatgaacgcttttcattggtgctcagtctttatgacagaggagcttgttgacaa ggataacaatttcta tgcggaactggaagagatttacgacgaaatctatccggtcatatccctgtataacctggt tcgcaactatgtcac gcaaaaaccatacagcacgaagaagattaaactgaactttggtattccgacgctggcccg cggatggtcaaaatc tgttgaatactcacgaaatgccataatcctgatgcgagataacctctactaccttggaat ctttaatgctaaaaa taaacccgataaaaaaattatcgaagggaacacgagtgaaaacaaaggtgattataaaaa aatgatatataatct gcttccaggaccaaataagatgatacccaaagttttcctttcttcaaagaccggcgtcga gacatataaaccatc cgcgtacatacttgaaggctacaaacaaaataaacatatcaaatcatctaaggattttga cattacgttctgtca tgatttgattgactatttcaaaaattgcatagccattcatccagagtggaaaaactttgg gtttgacttctctga taccagtacatatgaagacataagtggattttaccgagaagtagagctccaaggttataa aatagactggaccta tatatctgaaaaggatatagaccttttgcaagagaagggacagctttatcttttccaaat ctacaacaaagactt cagtaagaaaagtaccgggaatgacaatcttcataccatgtatctgaagaacctgttctc cgaagaaaatctgaa ggacatagtcctgaagcttaatggcgaagcggaaatttttttccgaaagagctctattaa gaaccccataataca taagaagggaagcattctcgttaatcgaacgtatgaggccgaagagaaagatcaatttgg gaatatccaaatcgt tcgaaagaacataccagaaaatatttaccaagaattgtacaaatattttaacgataaaag cgacaaagaactgtc tgatgaagctgctaagctgaaaaacgtcgtcggccatcatgaggccgcgacgaatatagt caaggattaccgata tacatacgataagtatttcctgcatatgcccatcactatcaactttaaggcaaataagac tggattcattaatga cagaatactgcaatacatagctaaagaaaaagatttgcatgttattggcattgacagggg tgagcgcaatcttat ctatgtaagcgtcattgatacttgcgggaatatcgtagagcagaagtcatttaatattgt aaatgggtacgatta ccaaatcaagttgaagcagcaagagggagcacgacagattgcccgcaaggagtggaaaga gatcggaaagataaa ggagatcaaggaggggtatttgtcccttgttatacacgaaatttccaagatggtaatcaa gtacaacgctataat tgctatggaggatctctcctatggatttaaaaagggaagatttaaagtcgagcggcaggt atatcagaaatttga aacaatgcttattaataaacttaattatctcgttttcaaagacattagtatcaccgaaaa cggtgggctgttgaa gggctatcaacttacgtacataccagataagcttaagaatgtgggtcaccaatgcggatg catattctacgtgcc cgcagcttatacaagcaaaatcgacccaacaacgggtttcgtaaacatatttaagttcaa ggatctcaccgtgga tgccaagcgagagttcataaaaaaatttgactcaatcagatatgactcagaaaagaatct tttttgttttacctt cgactacaataatttcattacacaaaatacggttatgagcaagtcatcctggtccgtata tacgtatggagtgcg cataaagcggagattcgttaacgggcgattttctaatgagtccgatacaatcgatataac aaaggatatggaaaa aactctggaaatgactgatataaattggagggacggtcatgacctcaggcaagacattat cgattatgagatcgt gcaacatatttttgagatctttcggttgactgtccaaatgaggaactctctgtctgaatt ggaagatagggacta cgatcgcctgataagccccgtgttgaacgagaataacatattctacgattccgcgaaagc cggggatgcgctccc taaggacgccgatgcaaatggggcctattgtattgctttgaaagggctgtacgaaatcaa acagatcaccgaaaa ctggaaagaagacgggaagtttagtcgggataaactgaagatatccaacaaggactggtt tgactttatccaaaa taagcgatatttgaaaaggccggcggccacgaaaaaggccggccaggcaaaaaagaaaaa gggatcctacccata cgatgttccagattacgcttatccctacgacgtgcctgattatgcatacccatatgatgt ccccgactatgccga gggcagaggaagtctgctaacatgcggtgacgtcgaggagaatcctggcccaatgaccga gtacaagcccacggt gcgcctcgccacccgcgacgacgtccccagggccgtacgcaccctcgccgccgcgttcgc cgactaccccgccac gcgccacaccgtcgatccggaccgccacatcgagcgggtcaccgagctgcaagaactctt cctcacgcgcgtcgg gctcgacatcggcaaggtgtgggtcgcggacgacggcgccgcggtggcggtctggaccac gccggagagcgtcga agcgggggcggtgttcgccgagatcggcccgcgcatggccgagttgagcggttcccggct ggccgcgcagcaaca gatggaaggcctcctggcgccgcaccggcccaaggagcccgcgtggttcctggccaccgt cggagtctcgcccga ccaccagggcaagggtctgggcagcgccgtcgtgctccccggagtggaggcggccgagcg cgccggggtgcccgc cttcctggagacctccgcgccccgcaacctccccttctacgagcggctcggcttcaccgt caccgccgacgtcga ggtgcccgaaggaccgcgcacctggtgcatgacccgcaagcccggtgcctgaactagtcc tgcaggcatgcaagc ttgatatcaagcttatcgataatcaacctctggattacaaaatttgtgaaagattgactg gtattcttaactatg ttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgctt cccgtatggctttca ttttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttg tcaggcaacgtggcg tggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtc agctcctttccggga ctttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgct gctggacaggggctc ggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggc tgctcgcctgtgttg ccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcgg accttccttcccgcg gcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcgga tctccctttgggccg cctccccgcatcgataccgtcgacctcgagggaattaattcgagctcggtacctttaaga ccgatgacttacaag gcagctgtagatcttagccactttttaaaagaaattaactgtgccttctagttgccagcc atctgttgtttgccc ctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaa tgaggaaattgcatc gcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaaggg ggaggattgggaaga gaatagcaggcatgctggggagcggccgcaggaacccctagtgatggagttggccactcc ctctctgcgcgctcg ctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggc ctcagtgagcgagcg agcgcgcagctgcctgcaggggcgcctgatgcggtattttctccttacgcatctgtgcgg tatttcacaccgcat acgtcaaagcaaccatagtacgcgccctgtagcggcgcattaagcgcggcgggtgtggtg gttacgcgcagcgtg accgctacacttgccagcgccttagcgcccgctcctttcgctttcttcccttcctttctc gccacgttcgccggc tttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacgg cacctcgaccccaaa aaacttgatttgggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgc cctttgacgttggag tccacgttctttaatagtggactcttgttccaaactggaacaacactcaactctatctcg ggctattcttttgat ttataagggattttgccgatttcggtctattggttaaaaaatgagctgatttaacaaaaa tttaacgcgaatttt aacaaaatattaacgtttacaattttatggtgcactctcagtacaatctgctctgatgcc gcatagttaagccag ccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatcc gcttacagacaagct gtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcg agacgaaagggcctc gtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggt ggcacttttcgggga aatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctc atgagacaataaccc tgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtc gcccttattcccttt tttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagat gctgaagatcagttg ggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagtttt cgccccgaagaacgt tttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgac gccgggcaagagcaa ctcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaa aagcatcttacggat ggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggcc aacttacttctgaca acgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaact cgccttgatcgttgg gaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagca atggcaacaacgttg cgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactgg atggaggcggataaa gttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatct ggagccggtgagcgt ggaagccgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagtt atctacacgacgggg agtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgatt aagcattggtaactg tcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaa aggatctaggtgaag atcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcg tcagaccccgtagaa aagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaaca aaaaaaccaccgcta ccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggc ttcagcagagcgcag ataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgta gcaccgcctacatac ctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttacc gggttggactcaaga cgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagccc agcttggagcgaacg acct acaccgaactgagat acct acagcgtgagct at gagaaagcgccacgcttcccgaagggagaaaggcggac aggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccaggggga aacgcctggtatctt tatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtca ggggggcggagccta tggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgct cacatgt

Construct E (SEQ ID NO:49) gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagag ataattggaattaat ttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttctt gggtagtttgcagtt ttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgat ttcttggctttatat atcttgtggaaaggacgaaacaccgggtcttcgagaagacctgttttagagctagaaata gcaagttaaaataag gctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcttttttgttttagagct agaaatagcaagtta aaataaggctagtccgtttttagcgcgtgcgccaattctgcagacaaatggctctagagg tacccgttacataac ttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaatag taacgccaataggga ctttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatc aagtgtatcatatgc caagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattgtgcccagt acatgaccttatggg actttcctacttggcagtacatctacgtattagtcatcgctattaccatggtcgaggtga gccccacgttctgct tcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaat tattttgtgcagcga tgggggcggggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggg gcggggcgaggcgga gaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggc ggcggcggcggcggc cctataaaaagcgaagcgcgcggcgggcgggagtcgctgcgcgctgccttcgccccgtgc cccgctccgccgccg cctcgcgccgcccgccccggctctgactgaccgcgttactcccacaggtgagcgggcggg acggcccttctcctc cgggctgtaattagctgagcaagaggtaagggtttaagggatggttggttggtggggtat taatgtttaattacc tggagcacctgcctgaaatcactttttttcaggttggaccggtgccaccatgacacagtt cgagggctttaccaa cctgtatcaggtgagcaagacactgcggtttgagctgatcccacagggcaagaccctgaa gcacatccaggagca gggcttcatcgaggaggacaaggcccgcaatgatcactacaaggagctgaagcccatcat cgatcggatctacaa gacctatgccgaccagtgcctgcagctggtgcagctggattgggagaacctgagcgccgc catcgactcctatag aaaggagaaaaccgaggagacaaggaacgccctgatcgaggagcaggccacatatcgcaa tgccatccacgacta cttcatcggccggacagacaacctgaccgatgccatcaataagagacacgccgagatcta caagggcctgttcaa ggccgagctgtttaatggcaaggtgctgaagcagctgggcaccgtgaccacaaccgagca cgagaacgccctgct gcggagcttcgacaagtttacaacctacttctccggcttttatagaaacaggaagaacgt gttcagcgccgagga tatcagcacagccatcccacaccgcatcgtgcaggacaacttccccaagtttaaggagaa ttgtcacatcttcac acgcctgatcaccgccgtgcccagcctgcgggagcactttgagaacgtgaagaaggccat cggcatcttcgtgag cacctccatcgaggaggtgttttccttccctttttataaccagctgctgacacagaccca gatcgacctgtataa ccagctgctgggaggaatctctcgggaggcaggcaccgagaagatcaagggcctgaacga ggtgctgaatctggc catccagaagaatgatgagacagcccacatcatcgcctccctgccacacagattcatccc cctgtttaagcagat cctgtccgataggaacaccctgtctttcatcctggaggagtttaagagcgacgaggaagt gatccagtccttctg caagtacaagacactgctgagaaacgagaacgtgctggagacagccgaggccctgtttaa cgagctgaacagcat cgacctgacacacatcttcatcagccacaagaagctggagacaatcagcagcgccctgtg cgaccactgggatac actgaggaatgccctgtatgagcggagaatctccgagctgacaggcaagatcaccaagtc tgccaaggagaaggt gcagcgcagcctgaagcacgaggatatcaacctgcaggagatcatctctgccgcaggcaa ggagctgagcgaggc cttcaagcagaaaaccagcgagatcctgtcccacgcacacgccgccctggatcagccact gcctacaaccctgaa gaagcaggaggagaaggagatcctgaagtctcagctggacagcctgctgggcctgtacca cctgctggactggtt tgccgtggatgagtccaacgaggtggaccccgagttctctgcccggctgaccggcatcaa gctggagatggagcc ttctctgagcttctacaacaaggccagaaattatgccaccaagaagccctactccgtgga gaagttcaagctgaa ctttcagatgcctacactggccagaggctgggacgtgaatgtggagaagaacagaggcgc catcctgtttgtgaa gaacggcctgtactatctgggcatcatgccaaagcagaagggcaggtataaggccctgag cttcgagcccacaga gaaaaccagcgagggctttgataagatgtactatgactacttccctgatgccgccaagat gatcccaaagtgcag cacccagctgaaggccgtgacagcccactttcagacccacacaacccccatcctgctgtc caacaatttcatcga gcctctggagatcacaaaggagatctacgacctgaacaatcctgagaaggagccaaagaa gtttcagacagccta cgccaagaaaaccggcgaccagaagggctacagagaggccctgtgcaagtggatcgactt cacaagggattttct gtccaagtataccaagacaacctctatcgatctgtctagcctgcggccatcctctcagta taaggacctgggcga gtactatgccgagctgaatcccctgctgtaccacatcagcttccagagaatcgccgagaa ggagatcatggatgc cgtggagacaggcaagctgtacctgttccagatctataacaaggactttgccaagggcca ccacggcaagcctaa tctgcacacactgtattggaccggcctgttttctccagagaacctggccaagacaagcat caagctgaatggcca ggccgagctgttctaccgccctaagtccaggatgaagaggatggcacaccggctgggaga gaagatgctgaacaa gaagctgaaggatcagaaaaccccaatccccgacaccctgtaccaggagctgtacgacta tgtgaatcacagact gtcccacgacctgtctgatgaggccagggccctgctgcccaacgtgatcaccaaggaggt gtctcacgagatcat caaggataggcgctttaccagcgacaagttctttttccacgtgcctatcacactgaacta tcaggccgccaattc cccatctaagttcaaccagagggtgaatgcctacctgaaggagcaccccgagacacctat catcggcatcgcccg gggcgagagaaacctgatctatatcacagtgatcgactccaccggcaagatcctggagca gcggagcctgaacac catccagcagtttgattaccagaagaagctggacaacagggagaaggagagggtggcagc aaggcaggcctggtc tgtggtgggcacaatcaaggatctgaagcagggctatctgagccaggtcatccacgagat cgtggacctgatgat ccactaccaggccgtggtggtgctggagaacctgaatttcggctttaagagcaagaggac cggcatcgccgagaa ggccgtgtaccagcagttcgagaagatgctgatcgataagctgaattgcctggtgctgaa ggactatccagcaga gaaagtgggaggcgtgctgaacccataccagctgacagaccagttcacctcctttgccaa gatgggcacccagtc tggcttcctgttttacgtgcctgccccatatacatctaagatcgatcccctgaccggctt cgtggaccccttcgt gtggaaaaccatcaagaatcacgagagccgcaagcacttcctggagggcttcgactttct gcactacgacgtgaa aaccggcgacttcatcctgcactttaagatgaacagaaatctgtccttccagaggggcct gcccggctttatgcc tgcatgggatatcgtgttcgagaagaacgagacacagtttgacgccaagggcaccccttt catcgccggcaagag aatcgtgccagtgatcgagaatcacagattcaccggcagataccgggacctgtatcctgc caacgagctgatcgc cctgctggaggagaagggcatcgtgttcagggatggctccaacatcctgccaaagctgct ggagaatgacgattc tcacgccatcgacaccatggtggccctgatccgcagcgtgctgcagatgcggaactccaa tgccgccacaggcga ggactatatcaacagccccgtgcgcgatctgaatggcgtgtgcttcgactcccggtttca gaacccagagtggcc catggacgccgatgccaatggcgcctaccacatcgccctgaagggccagctgctgctgaa tcacctgaaggagag caaggatctgaagctgcagaacggcatctccaatcaggactggctggcctacatccagga gctgcgcaacagcgg cagcgagactcccgggacctcagagtccgccacacccgaaagtcaactggtgaagagcga gctggaagagaagaa aagcgagctcagacataagctgaagtacgttccccacgaatacattgaactgatagaaat cgctagaaacagtac gcaagacagaatactggaaatgaaggtgatggagttcttcatgaaggtttacggctatcg tggcaaacacctcgg gggctcccggaagcccgacggggctatctacaccgtgggcagtcccatcgactatggcgt gatcgtggacaccaa agcttatagcggcggatataatctccccatcggccaagccgatgagatgcagaggtatgt ggaggagaaccaaac aagaaacaagcatatcaaccccaacgagtggtggaaggtttatcctagctcggtgaccga gtttaagttcctatt cgtgtctggccacttcaagggcaactataaggcacagctcactagactgaatcatatcac gaattgcaacggcgc cgtgttatccgtggaggagctactgatcggcggagagatgatcaaagccggcaccctgac cctggaagaggtgag aagaaagtttaacaatggcgaaataaatttcggcagcggaagtggaagcggctccatcac tagaaccaccaaccc tagaaacgtggtgcccaagatctacatgagcgccggcagcatccccctgaccacccacat caccaactcaattca gcccaccctgtggaccatcggcagcatcaacggcgtggcccccctggccaagagcatcaa gctgggcatccccgt gaccggcagcgcctacaccgatcagaccaccgccatggtgagaaagaaggtgagcgtgtt catgggcagcggcag cgggagcggctcatcgcagctggttaagagcgagttagaagaaaaaaagagcgaactgcg gcataaactgaagta tgtcccacacgagtacatcgaactgatcgagatcgcgagaaactctacccaagacagaat tctggagatgaaagt aatggaatttttcatgaaggtgtatggatatagagggaagcacctgggtggcagcagaaa acccgacggcgccat ctacactgtggggagccccatagactatggtgtgatcgtggataccaaggcgtatagcgg cggttacaatctgcc cattgggcaagcggacgagatgcaaagatatgtggaagagaatcagacgaggaacaagca cattaaccctaatga gtggtggaaggtctaccctagctccgttaccgagttcaagttcctgtttgtgagcgggca ttttaagggcaacta caaggcacagctgacccgcctgaaccacataacaaactgcaacggtgccgtgctgagcgt agaagagttgctaat cggcggcgagatgatcaaggccggcacgctaaccctcgaagaggtgcgcagaaagttcaa taacggcgaaatcaa tttcaaaaggccggcggccacgaaaaaggccggccaggcaaaaaagaaaaagggatccta cccatacgatgttcc agattacgcttatccctacgacgtgcctgattatgcatacccatatgatgtccccgacta tgccgagggcagagg aagtctgctaacatgcggtgacgtcgaggagaatcctggcccaatgaccgagtacaagcc cacggtgcgcctcgc cacccgcgacgacgtccccagggccgtacgcaccctcgccgccgcgttcgccgactaccc cgccacgcgccacac cgtcgatccggaccgccacatcgagcgggtcaccgagctgcaagaactcttcctcacgcg cgtcgggctcgacat cggcaaggtgtgggtcgcggacgacggcgccgcggtggcggtctggaccacgccggagag cgtcgaagcgggggc ggtgttcgccgagatcggcccgcgcatggccgagttgagcggttcccggctggccgcgca gcaacagatggaagg cctcctggcgccgcaccggcccaaggagcccgcgtggttcctggccaccgtcggagtctc gcccgaccaccaggg caagggtctgggcagcgccgtcgtgctccccggagtggaggcggccgagcgcgccggggt gcccgccttcctgga gacctccgcgccccgcaacctccccttctacgagcggctcggcttcaccgtcaccgccga cgtcgaggtgcccga aggaccgcgcacctggtgcatgacccgcaagcccggtgcctgaactagtcctgcaggcat gcaagcttgatatca agcttatcgataatcaacctctggattacaaaatttgtgaaagattgactggtattctta actatgttgctcctt ttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatgg ctttcattttctcct ccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaac gtggcgtggtgtgca ctgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctccttt ccgggactttcgctt tccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacag gggctcggctgttgg gcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcct gtgttgccacctgga ttctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttcctt cccgcggcctgctgc cggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctcccttt gggccgcctccccgc atcgataccgtcgacctcgagggaattaattcgagctcggtacctttaagaccgatgact tacaaggcagctgta gatcttagccactttttaaaagaaattaactgtgccttctagttgccagccatctgttgt ttgcccctcccccgt gccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaat tgcatcgcattgtct gagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattg ggaagagaatagcag gcatgctggggagcggccgcaggaacccctagtgatggagttggccactccctctctgcg cgctcgctcgctcac tgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgag cgagcgagcgcgcag ctgcctgcaggggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcaca ccgcatacgtcaaag caaccatagtacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgc agcgtgaccgctaca cttgccagcgccttagcgcccgctcctttcgctttcttcccttcctttctcgccacgttc gccggctttccccgt caagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgac cccaaaaaacttgat ttgggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacg ttggagtccacgttc tttaatagtggactcttgttccaaactggaacaacactcaactctatctcgggctattct tttgatttataaggg attttgccgatttcggtctattggttaaaaaatgagctgatttaacaaaaatttaacgcg aattttaacaaaata ttaacgtttacaattttatggtgcactctcagtacaatctgctctgatgccgcatagtta agccagccccgacac ccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacaga caagctgtgaccgtc tccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaag ggcctcgtgatacgc ctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcactttt cggggaaatgtgcgc ggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaa taaccctgataaatg cttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttatt cccttttttgcggca ttttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagat cagttgggtgcacga gtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaa gaacgttttccaatg atgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaa gagcaactcggtcgc cgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatctt acggatggcatgaca gtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttactt ctgacaacgatcgga ggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgat cgttgggaaccggag ctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaaca acgttgcgcaaacta ttaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcg gataaagttgcagga ccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggt gagcgtggaagccgc ggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacg acggggagtcaggca actatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattgg taactgtcagaccaa gtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctag gtgaagatccttttt gataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagacccc gtagaaaagatcaaa ggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaacca ccgctaccagcggtg gtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcaga gcgcagataccaaat actgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcct acatacctcgctctg ctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggac tcaagacgatagtta ccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggag cgaacgacctacacc gaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaag gcggacaggtatccg gtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctgg tatctttatagtcct gtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcgg agcctatggaaaaac gccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgt

Construct F (SEQ. ID NO:50) gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagag ataattggaattaat ttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttctt gggtagtttgcagtt ttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgat ttcttggctttatat atcttgtggaaaggacgaaacaccgggtcttcgagaagacctgttttagagctagaaata gcaagttaaaataag gctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcttttttgttttagagct agaaatagcaagtta aaataaggctagtccgtttttagcgcgtgcgccaattctgcagacaaatggctctagagg tacccgttacataac ttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaatag taacgccaataggga ctttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatc aagtgtatcatatgc caagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattgtgcccagt acatgaccttatggg actttcctacttggcagtacatctacgtattagtcatcgctattaccatggtcgaggtga gccccacgttctgct tcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaat tattttgtgcagcga tgggggcggggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggg gcggggcgaggcgga gaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggc ggcggcggcggcggc cctataaaaagcgaagcgcgcggcgggcgggagtcgctgcgcgctgccttcgccccgtgc cccgctccgccgccg cctcgcgccgcccgccccggctctgactgaccgcgttactcccacaggtgagcgggcggg acggcccttctcctc cgggctgtaattagctgagcaagaggtaagggtttaagggatggttggttggtggggtat taatgtttaattacc tggagcacctgcctgaaatcactttttttcaggttggaccggtgccaccatgcaactggt gaagagcgagctgga agagaagaaaagcgagctcagacataagctgaagtacgttccccacgaatacattgaact gatagaaatcgctag aaacagtacgcaagacagaatactggaaatgaaggtgatggagttcttcatgaaggttta cggctatcgtggcaa acacctcgggggctcccggaagcccgacggggctatctacaccgtgggcagtcccatcga ctatggcgtgatcgt ggacaccaaagcttatagcggcggatataatctccccatcggccaagccgatgagatgca gaggtatgtggagga gaaccaaacaagaaacaagcatatcaaccccaacgagtggtggaaggtttatcctagctc ggtgaccgagtttaa gttcctattcgtgtctggccacttcaagggcaactataaggcacagctcactagactgaa tcatatcacgaattg caacggcgccgtgttatccgtggaggagctactgatcggcggagagatgatcaaagccgg caccctgaccctgga agaggtgagaagaaagtttaacaatggcgaaataaatttcggcagcggaagtggaagcgg ctccatcactagaac caccaaccctagaaacgtggtgcccaagatctacatgagcgccggcagcatccccctgac cacccacatcaccaa ctcaattcagcccaccctgtggaccatcggcagcatcaacggcgtggcccccctggccaa gagcatcaagctggg catccccgtgaccggcagcgcctacaccgatcagaccaccgccatggtgagaaagaaggt gagcgtgttcatggg cagcggcagcgggagcggctcatcgcagctggttaagagcgagttagaagaaaaaaagag cgaactgcggcataa actgaagtatgtcccacacgagtacatcgaactgatcgagatcgcgagaaactctaccca agacagaattctgga gatgaaagtaatggaatttttcatgaaggtgtatggatatagagggaagcacctgggtgg cagcagaaaacccga cggcgccatctacactgtggggagccccatagactatggtgtgatcgtggataccaaggc gtatagcggcggtta caatctgcccattgggcaagcggacgagatgcaaagatatgtggaagagaatcagacgag gaacaagcacattaa ccctaatgagtggtggaaggtctaccctagctccgttaccgagttcaagttcctgtttgt gagcgggcattttaa gggcaactacaaggcacagctgacccgcctgaaccacataacaaactgcaacggtgccgt gctgagcgtagaaga gttgctaatcggcggcgagatgatcaaggccggcacgctaaccctcgaagaggtgcgcag aaagttcaataacgg cgaaatcaatttcagcggcagcgagactcccgggacctcagagtccgccacacccgaaag tacacagttcgaggg ctttaccaacctgtatcaggtgagcaagacactgcggtttgagctgatcccacagggcaa gaccctgaagcacat ccaggagcagggcttcatcgaggaggacaaggcccgcaatgatcactacaaggagctgaa gcccatcatcgatcg gatctacaagacctatgccgaccagtgcctgcagctggtgcagctggattgggagaacct gagcgccgccatcga ctcctatagaaaggagaaaaccgaggagacaaggaacgccctgatcgaggagcaggccac atatcgcaatgccat ccacgactacttcatcggccggacagacaacctgaccgatgccatcaataagagacacgc cgagatctacaaggg cctgttcaaggccgagctgtttaatggcaaggtgctgaagcagctgggcaccgtgaccac aaccgagcacgagaa cgccctgctgcggagcttcgacaagtttacaacctacttctccggcttttatagaaacag gaagaacgtgttcag cgccgaggatatcagcacagccatcccacaccgcatcgtgcaggacaacttccccaagtt taaggagaattgtca catcttcacacgcctgatcaccgccgtgcccagcctgcgggagcactttgagaacgtgaa gaaggccatcggcat cttcgtgagcacctccatcgaggaggtgttttccttccctttttataaccagctgctgac acagacccagatcga cctgtataaccagctgctgggaggaatctctcgggaggcaggcaccgagaagatcaaggg cctgaacgaggtgct gaatctggccatccagaagaatgatgagacagcccacatcatcgcctccctgccacacag attcatccccctgtt taagcagatcctgtccgataggaacaccctgtctttcatcctggaggagtttaagagcga cgaggaagtgatcca gtccttctgcaagtacaagacactgctgagaaacgagaacgtgctggagacagccgaggc cctgtttaacgagct gaacagcatcgacctgacacacatcttcatcagccacaagaagctggagacaatcagcag cgccctgtgcgacca ctgggatacactgaggaatgccctgtatgagcggagaatctccgagctgacaggcaagat caccaagtctgccaa ggagaaggtgcagcgcagcctgaagcacgaggatatcaacctgcaggagatcatctctgc cgcaggcaaggagct gagcgaggccttcaagcagaaaaccagcgagatcctgtcccacgcacacgccgccctgga tcagccactgcctac aaccctgaagaagcaggaggagaaggagatcctgaagtctcagctggacagcctgctggg cctgtaccacctgct ggactggtttgccgtggatgagtccaacgaggtggaccccgagttctctgcccggctgac cggcatcaagctgga gatggagccttctctgagcttctacaacaaggccagaaattatgccaccaagaagcccta ctccgtggagaagtt caagctgaactttcagatgcctacactggccagaggctgggacgtgaatgtggagaagaa cagaggcgccatcct gtttgtgaagaacggcctgtactatctgggcatcatgccaaagcagaagggcaggtataa ggccctgagcttcga gcccacagagaaaaccagcgagggctttgataagatgtactatgactacttccctgatgc cgccaagatgatccc aaagtgcagcacccagctgaaggccgtgacagcccactttcagacccacacaacccccat cctgctgtccaacaa tttcatcgagcctctggagatcacaaaggagatctacgacctgaacaatcctgagaagga gccaaagaagtttca gacagcctacgccaagaaaaccggcgaccagaagggctacagagaggccctgtgcaagtg gatcgacttcacaag ggattttctgtccaagtataccaagacaacctctatcgatctgtctagcctgcggccatc ctctcagtataagga cctgggcgagtactatgccgagctgaatcccctgctgtaccacatcagcttccagagaat cgccgagaaggagat catggatgccgtggagacaggcaagctgtacctgttccagatctataacaaggactttgc caagggccaccacgg caagcctaatctgcacacactgtattggaccggcctgttttctccagagaacctggccaa gacaagcatcaagct gaatggccaggccgagctgttctaccgccctaagtccaggatgaagaggatggcacaccg gctgggagagaagat gctgaacaagaagctgaaggatcagaaaaccccaatccccgacaccctgtaccaggagct gtacgactatgtgaa tcacagactgtcccacgacctgtctgatgaggccagggccctgctgcccaacgtgatcac caaggaggtgtctca cgagatcatcaaggataggcgctttaccagcgacaagttctttttccacgtgcctatcac actgaactatcaggc cgccaattccccatctaagttcaaccagagggtgaatgcctacctgaaggagcaccccga gacacctatcatcgg catcgcccggggcgagagaaacctgatctatatcacagtgatcgactccaccggcaagat cctggagcagcggag cctgaacaccatccagcagtttgattaccagaagaagctggacaacagggagaaggagag ggtggcagcaaggca ggcctggtctgtggtgggcacaatcaaggatctgaagcagggctatctgagccaggtcat ccacgagatcgtgga cctgatgatccactaccaggccgtggtggtgctggagaacctgaatttcggctttaagag caagaggaccggcat cgccgagaaggccgtgtaccagcagttcgagaagatgctgatcgataagctgaattgcct ggtgctgaaggacta tccagcagagaaagtgggaggcgtgctgaacccataccagctgacagaccagttcacctc ctttgccaagatggg cacccagtctggcttcctgttttacgtgcctgccccatatacatctaagatcgatcccct gaccggcttcgtgga ccccttcgtgtggaaaaccatcaagaatcacgagagccgcaagcacttcctggagggctt cgactttctgcacta cgacgtgaaaaccggcgacttcatcctgcactttaagatgaacagaaatctgtccttcca gaggggcctgcccgg ctttatgcctgcatgggatatcgtgttcgagaagaacgagacacagtttgacgccaaggg cacccctttcatcgc cggcaagagaatcgtgccagtgatcgagaatcacagattcaccggcagataccgggacct gtatcctgccaacga gctgatcgccctgctggaggagaagggcatcgtgttcagggatggctccaacatcctgcc aaagctgctggagaa tgacgattctcacgccatcgacaccatggtggccctgatccgcagcgtgctgcagatgcg gaactccaatgccgc cacaggcgaggactatatcaacagccccgtgcgcgatctgaatggcgtgtgcttcgactc ccggtttcagaaccc agagtggcccatggacgccgatgccaatggcgcctaccacatcgccctgaagggccagct gctgctgaatcacct gaaggagagcaaggatctgaagctgcagaacggcatctccaatcaggactggctggccta catccaggagctgcg caacaaaaggccggcggccacgaaaaaggccggccaggcaaaaaagaaaaagggatccta cccatacgatgttcc agattacgcttatccctacgacgtgcctgattatgcatacccatatgatgtccccgacta tgccgagggcagagg aagtctgctaacatgcggtgacgtcgaggagaatcctggcccaatgaccgagtacaagcc cacggtgcgcctcgc cacccgcgacgacgtccccagggccgtacgcaccctcgccgccgcgttcgccgactaccc cgccacgcgccacac cgtcgatccggaccgccacatcgagcgggtcaccgagctgcaagaactcttcctcacgcg cgtcgggctcgacat cggcaaggtgtgggtcgcggacgacggcgccgcggtggcggtctggaccacgccggagag cgtcgaagcgggggc ggtgttcgccgagatcggcccgcgcatggccgagttgagcggttcccggctggccgcgca gcaacagatggaagg cctcctggcgccgcaccggcccaaggagcccgcgtggttcctggccaccgtcggagtctc gcccgaccaccaggg caagggtctgggcagcgccgtcgtgctccccggagtggaggcggccgagcgcgccggggt gcccgccttcctgga gacctccgcgccccgcaacctccccttctacgagcggctcggcttcaccgtcaccgccga cgtcgaggtgcccga aggaccgcgcacctggtgcatgacccgcaagcccggtgcctgaactagtcctgcaggcat gcaagcttgatatca agcttatcgataatcaacctctggattacaaaatttgtgaaagattgactggtattctta actatgttgctcctt ttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatgg ctttcattttctcct ccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaac gtggcgtggtgtgca ctgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctccttt ccgggactttcgctt tccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacag gggctcggctgttgg gcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcct gtgttgccacctgga ttctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttcctt cccgcggcctgctgc cggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctcccttt gggccgcctccccgc atcgataccgtcgacctcgagggaattaattcgagctcggtacctttaagaccgatgact tacaaggcagctgta gatcttagccactttttaaaagaaattaactgtgccttctagttgccagccatctgttgt ttgcccctcccccgt gccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaat tgcatcgcattgtct gagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattg ggaagagaatagcag gcatgctggggagcggccgcaggaacccctagtgatggagttggccactccctctctgcg cgctcgctcgctcac tgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgag cgagcgagcgcgcag ctgcctgcaggggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcaca ccgcatacgtcaaag caaccatagtacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgc agcgtgaccgctaca cttgccagcgccttagcgcccgctcctttcgctttcttcccttcctttctcgccacgttc gccggctttccccgt caagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgac cccaaaaaacttgat ttgggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacg ttggagtccacgttc tttaatagtggactcttgttccaaactggaacaacactcaactctatctcgggctattct tttgatttataaggg attttgccgatttcggtctattggttaaaaaatgagctgatttaacaaaaatttaacgcg aattttaacaaaata ttaacgtttacaattttatggtgcactctcagtacaatctgctctgatgccgcatagtta agccagccccgacac ccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacaga caagctgtgaccgtc tccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaag ggcctcgtgatacgc ctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcactttt cggggaaatgtgcgc ggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaa taaccctgataaatg cttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttatt cccttttttgcggca ttttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagat cagttgggtgcacga gtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaa gaacgttttccaatg atgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaa gagcaactcggtcgc cgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatctt acggatggcatgaca gtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttactt ctgacaacgatcgga ggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgat cgttgggaaccggag ctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaaca acgttgcgcaaacta ttaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcg gataaagttgcagga ccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggt gagcgtggaagccgc ggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacg acggggagtcaggca actatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattgg taactgtcagaccaa gtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctag gtgaagatccttttt gataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagacccc gtagaaaagatcaaa ggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaacca ccgctaccagcggtg gtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcaga gcgcagataccaaat actgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcct acatacctcgctctg ctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggac tcaagacgatagtta ccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggag cgaacgacctacacc gaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaag gcggacaggtatccg gtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctgg tatctttatagtcct gtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcgg agcctatggaaaaac gccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgt

Construct G (SEQ ID NO:51) gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagag ataattggaattaat ttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttctt gggtagtttgcagtt ttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgat ttcttggctttatat atcttgtggaaaggacgaaacaccgggtcttcgagaagacctgttttagagctagaaata gcaagttaaaataag gctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcttttttgttttagagct agaaatagcaagtta aaataaggctagtccgtttttagcgcgtgcgccaattctgcagacaaatggctctagagg tacccgttacataac ttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaatag taacgccaataggga ctttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatc aagtgtatcatatgc caagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattgtgcccagt acatgaccttatggg actttcctacttggcagtacatctacgtattagtcatcgctattaccatggtcgaggtga gccccacgttctgct tcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaat tattttgtgcagcga tgggggcggggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggg gcggggcgaggcgga gaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggc ggcggcggcggcggc cctataaaaagcgaagcgcgcggcgggcgggagtcgctgcgcgctgccttcgccccgtgc cccgctccgccgccg cctcgcgccgcccgccccggctctgactgaccgcgttactcccacaggtgagcgggcggg acggcccttctcctc cgggctgtaattagctgagcaagaggtaagggtttaagggatggttggttggtggggtat taatgtttaattacc tggagcacctgcctgaaatcactttttttcaggttggaccggtgccaccatgacacagtt cgagggctttaccaa cctgtatcaggtgagcaagacactgcggtttgagctgatcccacagggcaagaccctgaa gcacatccaggagca gggcttcatcgaggaggacaaggcccgcaatgatcactacaaggagctgaagcccatcat cgatcggatctacaa gacctatgccgaccagtgcctgcagctggtgcagctggattgggagaacctgagcgccgc catcgactcctatag aaaggagaaaaccgaggagacaaggaacgccctgatcgaggagcaggccacatatcgcaa tgccatccacgacta cttcatcggccggacagacaacctgaccgatgccatcaataagagacacgccgagatcta caagggcctgttcaa ggccgagctgtttaatggcaaggtgctgaagcagctgggcaccgtgaccacaaccgagca cgagaacgccctgct gcggagcttcgacaagtttacaacctacttctccggcttttatagaaacaggaagaacgt gttcagcgccgagga tatcagcacagccatcccacaccgcatcgtgcaggacaacttccccaagtttaaggagaa ttgtcacatcttcac acgcctgatcaccgccgtgcccagcctgcgggagcactttgagaacgtgaagaaggccat cggcatcttcgtgag cacctccatcgaggaggtgttttccttccctttttataaccagctgctgacacagaccca gatcgacctgtataa ccagctgctgggaggaatctctcgggaggcaggcaccgagaagatcaagggcctgaacga ggtgctgaatctggc catccagaagaatgatgagacagcccacatcatcgcctccctgccacacagattcatccc cctgtttaagcagat cctgtccgataggaacaccctgtctttcatcctggaggagtttaagagcgacgaggaagt gatccagtccttctg caagtacaagacactgctgagaaacgagaacgtgctggagacagccgaggccctgtttaa cgagctgaacagcat cgacctgacacacatcttcatcagccacaagaagctggagacaatcagcagcgccctgtg cgaccactgggatac actgaggaatgccctgtatgagcggagaatctccgagctgacaggcaagatcaccaagtc tgccaaggagaaggt gcagcgcagcctgaagcacgaggatatcaacctgcaggagatcatctctgccgcaggcaa ggagctgagcgaggc cttcaagcagaaaaccagcgagatcctgtcccacgcacacgccgccctggatcagccact gcctacaaccctgaa gaagcaggaggagaaggagatcctgaagtctcagctggacagcctgctgggcctgtacca cctgctggactggtt tgccgtggatgagtccaacgaggtggaccccgagttctctgcccggctgaccggcatcaa gctggagatggagcc ttctctgagcttctacaacaaggccagaaattatgccaccaagaagccctactccgtgga gaagttcaagctgaa ctttcagatgcctacactggccagaggctgggacgtgaatgtggagaagaacagaggcgc catcctgtttgtgaa gaacggcctgtactatctgggcatcatgccaaagcagaagggcaggtataaggccctgag cttcgagcccacaga gaaaaccagcgagggctttgataagatgtactatgactacttccctgatgccgccaagat gatcccaaagtgcag cacccagctgaaggccgtgacagcccactttcagacccacacaacccccatcctgctgtc caacaatttcatcga gcctctggagatcacaaaggagatctacgacctgaacaatcctgagaaggagccaaagaa gtttcagacagccta cgccaagaaaaccggcgaccagaagggctacagagaggccctgtgcaagtggatcgactt cacaagggattttct gtccaagtataccaagacaacctctatcgatctgtctagcctgcggccatcctctcagta taaggacctgggcga gtactatgccgagctgaatcccctgctgtaccacatcagcttccagagaatcgccgagaa ggagatcatggatgc cgtggagacaggcaagctgtacctgttccagatctataacaaggactttgccaagggcca ccacggcaagcctaa tctgcacacactgtattggaccggcctgttttctccagagaacctggccaagacaagcat caagctgaatggcca ggccgagctgttctaccgccctaagtccaggatgaagaggatggcacaccggctgggaga gaagatgctgaacaa gaagctgaaggatcagaaaaccccaatccccgacaccctgtaccaggagctgtacgacta tgtgaatcacagact gtcccacgacctgtctgatgaggccagggccctgctgcccaacgtgatcaccaaggaggt gtctcacgagatcat caaggataggcgctttaccagcgacaagttctttttccacgtgcctatcacactgaacta tcaggccgccaattc cccatctaagttcaaccagagggtgaatgcctacctgaaggagcaccccgagacacctat catcggcatcgcccg gggcgagagaaacctgatctatatcacagtgatcgactccaccggcaagatcctggagca gcggagcctgaacac catccagcagtttgattaccagaagaagctggacaacagggagaaggagagggtggcagc aaggcaggcctggtc tgtggtgggcacaatcaaggatctgaagcagggctatctgagccaggtcatccacgagat cgtggacctgatgat ccactaccaggccgtggtggtgctggagaacctgaatttcggctttaagagcaagaggac cggcatcgccgagaa ggccgtgtaccagcagttcgagaagatgctgatcgataagctgaattgcctggtgctgaa ggactatccagcaga gaaagtgggaggcgtgctgaacccataccagctgacagaccagttcacctcctttgccaa gatgggcacccagtc tggcttcctgttttacgtgcctgccccatatacatctaagatcgatcccctgaccggctt cgtggaccccttcgt gtggaaaaccatcaagaatcacgagagccgcaagcacttcctggagggcttcgactttct gcactacgacgtgaa aaccggcgacttcatcctgcactttaagatgaacagaaatctgtccttccagaggggcct gcccggctttatgcc tgcatgggatatcgtgttcgagaagaacgagacacagtttgacgccaagggcaccccttt catcgccggcaagag aatcgtgccagtgatcgagaatcacagattcaccggcagataccgggacctgtatcctgc caacgagctgatcgc cctgctggaggagaagggcatcgtgttcagggatggctccaacatcctgccaaagctgct ggagaatgacgattc tcacgccatcgacaccatggtggccctgatccgcagcgtgctgcagatgcggaactccaa tgccgccacaggcga ggactatatcaacagccccgtgcgcgatctgaatggcgtgtgcttcgactcccggtttca gaacccagagtggcc catggacgccgatgccaatggcgcctaccacatcgccctgaagggccagctgctgctgaa tcacctgaaggagag caaggatctgaagctgcagaacggcatctccaatcaggactggctggcctacatccagga gctgcgcaacagcgg cagcgagactcccgggacctcagagtccgccacacccgaaagtcaactggtgaagagcga gctggaagagaagaa aagcgagctcagacataagctgaagtacgttccccacgaatacattgaactgatagaaat cgctagaaacagtac gcaagacagaatactggaaatgaaggtgatggagttcttcatgaaggtttacggctatcg tggcaaacacctcgg gggctcccggaagcccgccggggctatctacaccgtgggcagtcccatcgactatggcgt gatcgtggacaccaa agcttatagcggcggatataatctccccatcggccaagccgatgagatgcagaggtatgt ggaggagaaccaaac aagaaacaagcatatcaaccccaacgagtggtggaaggtttatcctagctcggtgaccga gtttaagttcctatt cgtgtctggccacttcaagggcaactataaggcacagctcactagactgaatcatatcac gaattgcaacggcgc cgtgttatccgtggaggagctactgatcggcggagagatgatcaaagccggcaccctgac cctggaagaggtgag aagaaagtttaacaatggcgaaataaatttcggcagcggaagtggaagcggctccatcac tagaaccaccaaccc tagaaacgtggtgcccaagatctacatgagcgccggcagcatccccctgaccacccacat caccaactcaattca gcccaccctgtggaccatcggcagcatcaacggcgtggcccccctggccaagagcatcaa gctgggcatccccgt gaccggcagcgcctacaccgatcagaccaccgccatggtgagaaagaaggtgagcgtgtt catgggcagcggcag cgggagcggctcatcgcagctggttaagagcgagttagaagaaaaaaagagcgaactgcg gcataaactgaagta tgtcccacacgagtacatcgaactgatcgagatcgcgagaaactctacccaagacagaat tctggagatgaaagt aatggaatttttcatgaaggtgtatggatatagagggaagcacctgggtggcagcagaaa acccgacggcgccat ctacactgtggggagccccatagactatggtgtgatcgtggataccaaggcgtatagcgg cggttacaatctgcc cattgggcaagcggacgagatgcaaagatatgtggaagagaatcagacgaggaacaagca cattaaccctaatga gtggtggaaggtctaccctagctccgttaccgagttcaagttcctgtttgtgagcgggca ttttaagggcaacta caaggcacagctgacccgcctgaaccacataacaaactgcaacggtgccgtgctgagcgt agaagagttgctaat cggcggcgagatgatcaaggccggcacgctaaccctcgaagaggtgcgcagaaagttcaa taacggcgaaatcaa tttcaaaaggccggcggccacgaaaaaggccggccaggcaaaaaagaaaaagggatccta cccatacgatgttcc agattacgcttatccctacgacgtgcctgattatgcatacccatatgatgtccccgacta tgccgagggcagagg aagtctgctaacatgcggtgacgtcgaggagaatcctggcccaatgaccgagtacaagcc cacggtgcgcctcgc cacccgcgacgacgtccccagggccgtacgcaccctcgccgccgcgttcgccgactaccc cgccacgcgccacac cgtcgatccggaccgccacatcgagcgggtcaccgagctgcaagaactcttcctcacgcg cgtcgggctcgacat cggcaaggtgtgggtcgcggacgacggcgccgcggtggcggtctggaccacgccggagag cgtcgaagcgggggc ggtgttcgccgagatcggcccgcgcatggccgagttgagcggttcccggctggccgcgca gcaacagatggaagg cctcctggcgccgcaccggcccaaggagcccgcgtggttcctggccaccgtcggagtctc gcccgaccaccaggg caagggtctgggcagcgccgtcgtgctccccggagtggaggcggccgagcgcgccggggt gcccgccttcctgga gacctccgcgccccgcaacctccccttctacgagcggctcggcttcaccgtcaccgccga cgtcgaggtgcccga aggaccgcgcacctggtgcatgacccgcaagcccggtgcctgaactagtcctgcaggcat gcaagcttgatatca agcttatcgataatcaacctctggattacaaaatttgtgaaagattgactggtattctta actatgttgctcctt ttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatgg ctttcattttctcct ccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaac gtggcgtggtgtgca ctgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctccttt ccgggactttcgctt tccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacag gggctcggctgttgg gcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcct gtgttgccacctgga ttctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttcctt cccgcggcctgctgc cggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctcccttt gggccgcctccccgc atcgataccgtcgacctcgagggaattaattcgagctcggtacctttaagaccgatgact tacaaggcagctgta gatcttagccactttttaaaagaaattaactgtgccttctagttgccagccatctgttgt ttgcccctcccccgt gccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaat tgcatcgcattgtct gagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattg ggaagagaatagcag gcatgctggggagcggccgcaggaacccctagtgatggagttggccactccctctctgcg cgctcgctcgctcac tgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgag cgagcgagcgcgcag ctgcctgcaggggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcaca ccgcatacgtcaaag caaccatagtacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgc agcgtgaccgctaca cttgccagcgccttagcgcccgctcctttcgctttcttcccttcctttctcgccacgttc gccggctttccccgt caagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgac cccaaaaaacttgat ttgggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacg ttggagtccacgttc tttaatagtggactcttgttccaaactggaacaacactcaactctatctcgggctattct tttgatttataaggg attttgccgatttcggtctattggttaaaaaatgagctgatttaacaaaaatttaacgcg aattttaacaaaata ttaacgtttacaattttatggtgcactctcagtacaatctgctctgatgccgcatagtta agccagccccgacac ccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacaga caagctgtgaccgtc tccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaag ggcctcgtgatacgc ctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcactttt cggggaaatgtgcgc ggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaa taaccctgataaatg cttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttatt cccttttttgcggca ttttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagat cagttgggtgcacga gtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaa gaacgttttccaatg atgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaa gagcaactcggtcgc cgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatctt acggatggcatgaca gtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttactt ctgacaacgatcgga ggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgat cgttgggaaccggag ctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaaca acgttgcgcaaacta ttaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcg gataaagttgcagga ccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggt gagcgtggaagccgc ggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacg acggggagtcaggca actatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattgg taactgtcagaccaa gtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctag gtgaagatccttttt gataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagacccc gtagaaaagatcaaa ggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaacca ccgctaccagcggtg gtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcaga gcgcagataccaaat actgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcct acatacctcgctctg ctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggac tcaagacgatagtta ccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggag cgaacgacctacacc gaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaag gcggacaggtatccg gtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctgg tatctttatagtcct gtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcgg agcctatggaaaaac gccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgt

Construct H (SEQ. ID NO:52) gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagag ataattggaattaat ttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttctt gggtagtttgcagtt ttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgat ttcttggctttatat atcttgtggaaaggacgaaacaccgggtcttcgagaagacctgttttagagctagaaata gcaagttaaaataag gctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcttttttgttttagagct agaaatagcaagtta aaataaggctagtccgtttttagcgcgtgcgccaattctgcagacaaatggctctagagg tacccgttacataac ttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaatag taacgccaataggga ctttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatc aagtgtatcatatgc caagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattgtgcccagt acatgaccttatggg actttcctacttggcagtacatctacgtattagtcatcgctattaccatggtcgaggtga gccccacgttctgct tcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaat tattttgtgcagcga tgggggcggggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggg gcggggcgaggcgga gaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggc ggcggcggcggcggc cctataaaaagcgaagcgcgcggcgggcgggagtcgctgcgcgctgccttcgccccgtgc cccgctccgccgccg cctcgcgccgcccgccccggctctgactgaccgcgttactcccacaggtgagcgggcggg acggcccttctcctc cgggctgtaattagctgagcaagaggtaagggtttaagggatggttggttggtggggtat taatgtttaattacc tggagcacctgcctgaaatcactttttttcaggttggaccggtgccaccatgacacagtt cgagggctttaccaa cctgtatcaggtgagcaagacactgcggtttgagctgatcccacagggcaagaccctgaa gcacatccaggagca gggcttcatcgaggaggacaaggcccgcaatgatcactacaaggagctgaagcccatcat cgatcggatctacaa gacctatgccgaccagtgcctgcagctggtgcagctggattgggagaacctgagcgccgc catcgactcctatag aaaggagaaaaccgaggagacaaggaacgccctgatcgaggagcaggccacatatcgcaa tgccatccacgacta cttcatcggccggacagacaacctgaccgatgccatcaataagagacacgccgagatcta caagggcctgttcaa ggccgagctgtttaatggcaaggtgctgaagcagctgggcaccgtgaccacaaccgagca cgagaacgccctgct gcggagcttcgacaagtttacaacctacttctccggcttttatagaaacaggaagaacgt gttcagcgccgagga tatcagcacagccatcccacaccgcatcgtgcaggacaacttccccaagtttaaggagaa ttgtcacatcttcac acgcctgatcaccgccgtgcccagcctgcgggagcactttgagaacgtgaagaaggccat cggcatcttcgtgag cacctccatcgaggaggtgttttccttccctttttataaccagctgctgacacagaccca gatcgacctgtataa ccagctgctgggaggaatctctcgggaggcaggcaccgagaagatcaagggcctgaacga ggtgctgaatctggc catccagaagaatgatgagacagcccacatcatcgcctccctgccacacagattcatccc cctgtttaagcagat cctgtccgataggaacaccctgtctttcatcctggaggagtttaagagcgacgaggaagt gatccagtccttctg caagtacaagacactgctgagaaacgagaacgtgctggagacagccgaggccctgtttaa cgagctgaacagcat cgacctgacacacatcttcatcagccacaagaagctggagacaatcagcagcgccctgtg cgaccactgggatac actgaggaatgccctgtatgagcggagaatctccgagctgacaggcaagatcaccaagtc tgccaaggagaaggt gcagcgcagcctgaagcacgaggatatcaacctgcaggagatcatctctgccgcaggcaa ggagctgagcgaggc cttcaagcagaaaaccagcgagatcctgtcccacgcacacgccgccctggatcagccact gcctacaaccctgaa gaagcaggaggagaaggagatcctgaagtctcagctggacagcctgctgggcctgtacca cctgctggactggtt tgccgtggatgagtccaacgaggtggaccccgagttctctgcccggctgaccggcatcaa gctggagatggagcc ttctctgagcttctacaacaaggccagaaattatgccaccaagaagccctactccgtgga gaagttcaagctgaa ctttcagatgcctacactggccagaggctgggacgtgaatgtggagaagaacagaggcgc catcctgtttgtgaa gaacggcctgtactatctgggcatcatgccaaagcagaagggcaggtataaggccctgag cttcgagcccacaga gaaaaccagcgagggctttgataagatgtactatgactacttccctgatgccgccaagat gatcccaaagtgcag cacccagctgaaggccgtgacagcccactttcagacccacacaacccccatcctgctgtc caacaatttcatcga gcctctggagatcacaaaggagatctacgacctgaacaatcctgagaaggagccaaagaa gtttcagacagccta cgccaagaaaaccggcgaccagaagggctacagagaggccctgtgcaagtggatcgactt cacaagggattttct gtccaagtataccaagacaacctctatcgatctgtctagcctgcggccatcctctcagta taaggacctgggcga gtactatgccgagctgaatcccctgctgtaccacatcagcttccagagaatcgccgagaa ggagatcatggatgc cgtggagacaggcaagctgtacctgttccagatctataacaaggactttgccaagggcca ccacggcaagcctaa tctgcacacactgtattggaccggcctgttttctccagagaacctggccaagacaagcat caagctgaatggcca ggccgagctgttctaccgccctaagtccaggatgaagaggatggcacaccggctgggaga gaagatgctgaacaa gaagctgaaggatcagaaaaccccaatccccgacaccctgtaccaggagctgtacgacta tgtgaatcacagact gtcccacgacctgtctgatgaggccagggccctgctgcccaacgtgatcaccaaggaggt gtctcacgagatcat caaggataggcgctttaccagcgacaagttctttttccacgtgcctatcacactgaacta tcaggccgccaattc cccatctaagttcaaccagagggtgaatgcctacctgaaggagcaccccgagacacctat catcggcatcgcccg gggcgagagaaacctgatctatatcacagtgatcgactccaccggcaagatcctggagca gcggagcctgaacac catccagcagtttgattaccagaagaagctggacaacagggagaaggagagggtggcagc aaggcaggcctggtc tgtggtgggcacaatcaaggatctgaagcagggctatctgagccaggtcatccacgagat cgtggacctgatgat ccactaccaggccgtggtggtgctggagaacctgaatttcggctttaagagcaagaggac cggcatcgccgagaa ggccgtgtaccagcagttcgagaagatgctgatcgataagctgaattgcctggtgctgaa ggactatccagcaga gaaagtgggaggcgtgctgaacccataccagctgacagaccagttcacctcctttgccaa gatgggcacccagtc tggcttcctgttttacgtgcctgccccatatacatctaagatcgatcccctgaccggctt cgtggaccccttcgt gtggaaaaccatcaagaatcacgagagccgcaagcacttcctggagggcttcgactttct gcactacgacgtgaa aaccggcgacttcatcctgcactttaagatgaacagaaatctgtccttccagaggggcct gcccggctttatgcc tgcatgggatatcgtgttcgagaagaacgagacacagtttgacgccaagggcaccccttt catcgccggcaagag aatcgtgccagtgatcgagaatcacagattcaccggcagataccgggacctgtatcctgc caacgagctgatcgc cctgctggaggagaagggcatcgtgttcagggatggctccaacatcctgccaaagctgct ggagaatgacgattc tcacgccatcgacaccatggtggccctgatccgcagcgtgctgcagatgcggaactccaa tgccgccacaggcga ggactatatcaacagccccgtgcgcgatctgaatggcgtgtgcttcgactcccggtttca gaacccagagtggcc catggacgccgatgccaatggcgcctaccacatcgccctgaagggccagctgctgctgaa tcacctgaaggagag caaggatctgaagctgcagaacggcatctccaatcaggactggctggcctacatccagga gctgcgcaacagcgg cagcgagactcccgggacctcagagtccgccacacccgaaagtcaactggtgaagagcga gctggaagagaagaa aagcgagctcagacataagctgaagtacgttccccacgaatacattgaactgatagaaat cgctagaaacagtac gcaagacagaatactggaaatgaaggtgatggagttcttcatgaaggtttacggctatcg tggcaaacacctcgg gggctcccggaagcccgacggggctatctacaccgtgggcagtcccatcgactatggcgt gatcgtggacaccaa agcttatagcggcggatataatctccccatcggccaagccgatgagatgcagaggtatgt ggaggagaaccaaac aagaaacaagcatatcaaccccaacgagtggtggaaggtttatcctagctcggtgaccga gtttaagttcctatt cgtgtctggccacttcaagggcaactataaggcacagctcactagactgaatcatatcac gaattgcaacggcgc cgtgttatccgtggaggagctactgatcggcggagagatgatcaaagccggcaccctgac cctggaagaggtgag aagaaagtttaacaatggcgaaataaatttcggcagcggaagtggaagcggctccatcac tagaaccaccaaccc tagaaacgtggtgcccaagatctacatgagcgccggcagcatccccctgaccacccacat caccaactcaattca gcccaccctgtggaccatcggcagcatcaacggcgtggcccccctggccaagagcatcaa gctgggcatccccgt gaccggcagcgcctacaccgatcagaccaccgccatggtgagaaagaaggtgagcgtgtt catgggcagcggcag cgggagcggctcatcgcagctggttaagagcgagttagaagaaaaaaagagcgaactgcg gcataaactgaagta tgtcccacacgagtacatcgaactgatcgagatcgcgagaaactctacccaagacagaat tctggagatgaaagt aatggaatttttcatgaaggtgtatggatatagagggaagcacctgggtggcagcagaaa acccgccggcgccat ctacactgtggggagccccatagactatggtgtgatcgtggataccaaggcgtatagcgg cggttacaatctgcc cattgggcaagcggacgagatgcaaagatatgtggaagagaatcagacgaggaacaagca cattaaccctaatga gtggtggaaggtctaccctagctccgttaccgagttcaagttcctgtttgtgagcgggca ttttaagggcaacta caaggcacagctgacccgcctgaaccacataacaaactgcaacggtgccgtgctgagcgt agaagagttgctaat cggcggcgagatgatcaaggccggcacgctaaccctcgaagaggtgcgcagaaagttcaa taacggcgaaatcaa tttcaaaaggccggcggccacgaaaaaggccggccaggcaaaaaagaaaaagggatccta cccatacgatgttcc agattacgcttatccctacgacgtgcctgattatgcatacccatatgatgtccccgacta tgccgagggcagagg aagtctgctaacatgcggtgacgtcgaggagaatcctggcccaatgaccgagtacaagcc cacggtgcgcctcgc cacccgcgacgacgtccccagggccgtacgcaccctcgccgccgcgttcgccgactaccc cgccacgcgccacac cgtcgatccggaccgccacatcgagcgggtcaccgagctgcaagaactcttcctcacgcg cgtcgggctcgacat cggcaaggtgtgggtcgcggacgacggcgccgcggtggcggtctggaccacgccggagag cgtcgaagcgggggc ggtgttcgccgagatcggcccgcgcatggccgagttgagcggttcccggctggccgcgca gcaacagatggaagg cctcctggcgccgcaccggcccaaggagcccgcgtggttcctggccaccgtcggagtctc gcccgaccaccaggg caagggtctgggcagcgccgtcgtgctccccggagtggaggcggccgagcgcgccggggt gcccgccttcctgga gacctccgcgccccgcaacctccccttctacgagcggctcggcttcaccgtcaccgccga cgtcgaggtgcccga aggaccgcgcacctggtgcatgacccgcaagcccggtgcctgaactagtcctgcaggcat gcaagcttgatatca agcttatcgataatcaacctctggattacaaaatttgtgaaagattgactggtattctta actatgttgctcctt ttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatgg ctttcattttctcct ccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaac gtggcgtggtgtgca ctgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctccttt ccgggactttcgctt tccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacag gggctcggctgttgg gcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcct gtgttgccacctgga ttctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttcctt cccgcggcctgctgc cggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctcccttt gggccgcctccccgc atcgataccgtcgacctcgagggaattaattcgagctcggtacctttaagaccgatgact tacaaggcagctgta gatcttagccactttttaaaagaaattaactgtgccttctagttgccagccatctgttgt ttgcccctcccccgt gccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaat tgcatcgcattgtct gagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattg ggaagagaatagcag gcatgctggggagcggccgcaggaacccctagtgatggagttggccactccctctctgcg cgctcgctcgctcac tgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgag cgagcgagcgcgcag ctgcctgcaggggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcaca ccgcatacgtcaaag caaccatagtacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgc agcgtgaccgctaca cttgccagcgccttagcgcccgctcctttcgctttcttcccttcctttctcgccacgttc gccggctttccccgt caagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgac cccaaaaaacttgat ttgggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacg ttggagtccacgttc tttaatagtggactcttgttccaaactggaacaacactcaactctatctcgggctattct tttgatttataaggg attttgccgatttcggtctattggttaaaaaatgagctgatttaacaaaaatttaacgcg aattttaacaaaata ttaacgtttacaattttatggtgcactctcagtacaatctgctctgatgccgcatagtta agccagccccgacac ccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacaga caagctgtgaccgtc tccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaag ggcctcgtgatacgc ctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcactttt cggggaaatgtgcgc ggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaa taaccctgataaatg cttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttatt cccttttttgcggca ttttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagat cagttgggtgcacga gtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaa gaacgttttccaatg atgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaa gagcaactcggtcgc cgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatctt acggatggcatgaca gtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttactt ctgacaacgatcgga ggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgat cgttgggaaccggag ctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaaca acgttgcgcaaacta ttaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcg gataaagttgcagga ccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggt gagcgtggaagccgc ggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacg acggggagtcaggca actatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattgg taactgtcagaccaa gtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctag gtgaagatccttttt gataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagacccc gtagaaaagatcaaa ggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaacca ccgctaccagcggtg gtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcaga gcgcagataccaaat act gttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctaca t acct cgctctg ctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggac tcaagacgatagtta ccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggag cgaacgacctacacc gaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaag gcggacaggtatccg gtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctgg tatctttatagtcct gtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcgg agcctatggaaaaac gccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgt

Construct ! (SEQ ID NO:53) gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagag ataattggaattaat ttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttctt gggtagtttgcagtt ttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgat ttcttggctttatat atcttgtggaaaggacgaaacaccgggtcttcgagaagacctgttttagagctagaaata gcaagttaaaataag gctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcttttttgttttagagct agaaatagcaagtta aaataaggctagtccgtttttagcgcgtgcgccaattctgcagacaaatggctctagagg tacccgttacataac ttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaatag taacgccaataggga ctttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatc aagtgtatcatatgc caagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattgtgcccagt acatgaccttatggg actttcctacttggcagtacatctacgtattagtcatcgctattaccatggtcgaggtga gccccacgttctgct tcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaat tattttgtgcagcga tgggggcggggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggg gcggggcgaggcgga gaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggc ggcggcggcggcggc cctataaaaagcgaagcgcgcggcgggcgggagtcgctgcgcgctgccttcgccccgtgc cccgctccgccgccg cctcgcgccgcccgccccggctctgactgaccgcgttactcccacaggtgagcgggcggg acggcccttctcctc cgggctgtaattagctgagcaagaggtaagggtttaagggatggttggttggtggggtat taatgtttaattacc tggagcacctgcctgaaatcactttttttcaggttggaccggtgccaccatgcaactggt gaagagcgagctgga agagaagaaaagcgagctcagacataagctgaagtacgttccccacgaatacattgaact gatagaaatcgctag aaacagtacgcaagacagaatactggaaatgaaggtgatggagttcttcatgaaggttta cggctatcgtggcaa acacctcgggggctcccggaagcccgccggggctatctacaccgtgggcagtcccatcga ctatggcgtgatcgt ggacaccaaagcttatagcggcggatataatctccccatcggccaagccgatgagatgca gaggtatgtggagga gaaccaaacaagaaacaagcatatcaaccccaacgagtggtggaaggtttatcctagctc ggtgaccgagtttaa gttcctattcgtgtctggccacttcaagggcaactataaggcacagctcactagactgaa tcatatcacgaattg caacggcgccgtgttatccgtggaggagctactgatcggcggagagatgatcaaagccgg caccctgaccctgga agaggtgagaagaaagtttaacaatggcgaaataaatttcggcagcggaagtggaagcgg ctccatcactagaac caccaaccctagaaacgtggtgcccaagatctacatgagcgccggcagcatccccctgac cacccacatcaccaa ctcaattcagcccaccctgtggaccatcggcagcatcaacggcgtggcccccctggccaa gagcatcaagctggg catccccgtgaccggcagcgcctacaccgatcagaccaccgccatggtgagaaagaaggt gagcgtgttcatggg cagcggcagcgggagcggctcatcgcagctggttaagagcgagttagaagaaaaaaagag cgaactgcggcataa actgaagtatgtcccacacgagtacatcgaactgatcgagatcgcgagaaactctaccca agacagaattctgga gatgaaagtaatggaatttttcatgaaggtgtatggatatagagggaagcacctgggtgg cagcagaaaacccga cggcgccatctacactgtggggagccccatagactatggtgtgatcgtggataccaaggc gtatagcggcggtta caatctgcccattgggcaagcggacgagatgcaaagatatgtggaagagaatcagacgag gaacaagcacattaa ccctaatgagtggtggaaggtctaccctagctccgttaccgagttcaagttcctgtttgt gagcgggcattttaa gggcaactacaaggcacagctgacccgcctgaaccacataacaaactgcaacggtgccgt gctgagcgtagaaga gttgctaatcggcggcgagatgatcaaggccggcacgctaaccctcgaagaggtgcgcag aaagttcaataacgg cgaaatcaatttcagcggcagcgagactcccgggacctcagagtccgccacacccgaaag tacacagttcgaggg ctttaccaacctgtatcaggtgagcaagacactgcggtttgagctgatcccacagggcaa gaccctgaagcacat ccaggagcagggcttcatcgaggaggacaaggcccgcaatgatcactacaaggagctgaa gcccatcatcgatcg gatctacaagacctatgccgaccagtgcctgcagctggtgcagctggattgggagaacct gagcgccgccatcga ctcctatagaaaggagaaaaccgaggagacaaggaacgccctgatcgaggagcaggccac atatcgcaatgccat ccacgactacttcatcggccggacagacaacctgaccgatgccatcaataagagacacgc cgagatctacaaggg cctgttcaaggccgagctgtttaatggcaaggtgctgaagcagctgggcaccgtgaccac aaccgagcacgagaa cgccctgctgcggagcttcgacaagtttacaacctacttctccggcttttatagaaacag gaagaacgtgttcag cgccgaggatatcagcacagccatcccacaccgcatcgtgcaggacaacttccccaagtt taaggagaattgtca catcttcacacgcctgatcaccgccgtgcccagcctgcgggagcactttgagaacgtgaa gaaggccatcggcat cttcgtgagcacctccatcgaggaggtgttttccttccctttttataaccagctgctgac acagacccagatcga cctgtataaccagctgctgggaggaatctctcgggaggcaggcaccgagaagatcaaggg cctgaacgaggtgct gaatctggccatccagaagaatgatgagacagcccacatcatcgcctccctgccacacag attcatccccctgtt taagcagatcctgtccgataggaacaccctgtctttcatcctggaggagtttaagagcga cgaggaagtgatcca gtccttctgcaagtacaagacactgctgagaaacgagaacgtgctggagacagccgaggc cctgtttaacgagct gaacagcatcgacctgacacacatcttcatcagccacaagaagctggagacaatcagcag cgccctgtgcgacca ctgggatacactgaggaatgccctgtatgagcggagaatctccgagctgacaggcaagat caccaagtctgccaa ggagaaggtgcagcgcagcctgaagcacgaggatatcaacctgcaggagatcatctctgc cgcaggcaaggagct gagcgaggccttcaagcagaaaaccagcgagatcctgtcccacgcacacgccgccctgga tcagccactgcctac aaccctgaagaagcaggaggagaaggagatcctgaagtctcagctggacagcctgctggg cctgtaccacctgct ggactggtttgccgtggatgagtccaacgaggtggaccccgagttctctgcccggctgac cggcatcaagctgga gatggagccttctctgagcttctacaacaaggccagaaattatgccaccaagaagcccta ctccgtggagaagtt caagctgaactttcagatgcctacactggccagaggctgggacgtgaatgtggagaagaa cagaggcgccatcct gtttgtgaagaacggcctgtactatctgggcatcatgccaaagcagaagggcaggtataa ggccctgagcttcga gcccacagagaaaaccagcgagggctttgataagatgtactatgactacttccctgatgc cgccaagatgatccc aaagtgcagcacccagctgaaggccgtgacagcccactttcagacccacacaacccccat cctgctgtccaacaa tttcatcgagcctctggagatcacaaaggagatctacgacctgaacaatcctgagaagga gccaaagaagtttca gacagcctacgccaagaaaaccggcgaccagaagggctacagagaggccctgtgcaagtg gatcgacttcacaag ggattttctgtccaagtataccaagacaacctctatcgatctgtctagcctgcggccatc ctctcagtataagga cctgggcgagtactatgccgagctgaatcccctgctgtaccacatcagcttccagagaat cgccgagaaggagat catggatgccgtggagacaggcaagctgtacctgttccagatctataacaaggactttgc caagggccaccacgg caagcctaatctgcacacactgtattggaccggcctgttttctccagagaacctggccaa gacaagcatcaagct gaatggccaggccgagctgttctaccgccctaagtccaggatgaagaggatggcacaccg gctgggagagaagat gctgaacaagaagctgaaggatcagaaaaccccaatccccgacaccctgtaccaggagct gtacgactatgtgaa tcacagactgtcccacgacctgtctgatgaggccagggccctgctgcccaacgtgatcac caaggaggtgtctca cgagatcatcaaggataggcgctttaccagcgacaagttctttttccacgtgcctatcac actgaactatcaggc cgccaattccccatctaagttcaaccagagggtgaatgcctacctgaaggagcaccccga gacacctatcatcgg catcgcccggggcgagagaaacctgatctatatcacagtgatcgactccaccggcaagat cctggagcagcggag cctgaacaccatccagcagtttgattaccagaagaagctggacaacagggagaaggagag ggtggcagcaaggca ggcctggtctgtggtgggcacaatcaaggatctgaagcagggctatctgagccaggtcat ccacgagatcgtgga cctgatgatccactaccaggccgtggtggtgctggagaacctgaatttcggctttaagag caagaggaccggcat cgccgagaaggccgtgtaccagcagttcgagaagatgctgatcgataagctgaattgcct ggtgctgaaggacta tccagcagagaaagtgggaggcgtgctgaacccataccagctgacagaccagttcacctc ctttgccaagatggg cacccagtctggcttcctgttttacgtgcctgccccatatacatctaagatcgatcccct gaccggcttcgtgga ccccttcgtgtggaaaaccatcaagaatcacgagagccgcaagcacttcctggagggctt cgactttctgcacta cgacgtgaaaaccggcgacttcatcctgcactttaagatgaacagaaatctgtccttcca gaggggcctgcccgg ctttatgcctgcatgggatatcgtgttcgagaagaacgagacacagtttgacgccaaggg cacccctttcatcgc cggcaagagaatcgtgccagtgatcgagaatcacagattcaccggcagataccgggacct gtatcctgccaacga gctgatcgccctgctggaggagaagggcatcgtgttcagggatggctccaacatcctgcc aaagctgctggagaa tgacgattctcacgccatcgacaccatggtggccctgatccgcagcgtgctgcagatgcg gaactccaatgccgc cacaggcgaggactatatcaacagccccgtgcgcgatctgaatggcgtgtgcttcgactc ccggtttcagaaccc agagtggcccatggacgccgatgccaatggcgcctaccacatcgccctgaagggccagct gctgctgaatcacct gaaggagagcaaggatctgaagctgcagaacggcatctccaatcaggactggctggccta catccaggagctgcg caacaaaaggccggcggccacgaaaaaggccggccaggcaaaaaagaaaaagggatccta cccatacgatgttcc agattacgcttatccctacgacgtgcctgattatgcatacccatatgatgtccccgacta tgccgagggcagagg aagtctgctaacatgcggtgacgtcgaggagaatcctggcccaatgaccgagtacaagcc cacggtgcgcctcgc cacccgcgacgacgtccccagggccgtacgcaccctcgccgccgcgttcgccgactaccc cgccacgcgccacac cgtcgatccggaccgccacatcgagcgggtcaccgagctgcaagaactcttcctcacgcg cgtcgggctcgacat cggcaaggtgtgggtcgcggacgacggcgccgcggtggcggtctggaccacgccggagag cgtcgaagcgggggc ggtgttcgccgagatcggcccgcgcatggccgagttgagcggttcccggctggccgcgca gcaacagatggaagg cctcctggcgccgcaccggcccaaggagcccgcgtggttcctggccaccgtcggagtctc gcccgaccaccaggg caagggtctgggcagcgccgtcgtgctccccggagtggaggcggccgagcgcgccggggt gcccgccttcctgga gacctccgcgccccgcaacctccccttctacgagcggctcggcttcaccgtcaccgccga cgtcgaggtgcccga aggaccgcgcacctggtgcatgacccgcaagcccggtgcctgaactagtcctgcaggcat gcaagcttgatatca agcttatcgataatcaacctctggattacaaaatttgtgaaagattgactggtattctta actatgttgctcctt ttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatgg ctttcattttctcct ccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaac gtggcgtggtgtgca ctgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctccttt ccgggactttcgctt tccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacag gggctcggctgttgg gcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcct gtgttgccacctgga ttctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttcctt cccgcggcctgctgc cggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctcccttt gggccgcctccccgc atcgataccgtcgacctcgagggaattaattcgagctcggtacctttaagaccgatgact tacaaggcagctgta gatcttagccactttttaaaagaaattaactgtgccttctagttgccagccatctgttgt ttgcccctcccccgt gccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaat tgcatcgcattgtct gagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattg ggaagagaatagcag gcatgctggggagcggccgcaggaacccctagtgatggagttggccactccctctctgcg cgctcgctcgctcac tgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgag cgagcgagcgcgcag ctgcctgcaggggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcaca ccgcatacgtcaaag caaccatagtacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgc agcgtgaccgctaca cttgccagcgccttagcgcccgctcctttcgctttcttcccttcctttctcgccacgttc gccggctttccccgt caagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgac cccaaaaaacttgat ttgggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacg ttggagtccacgttc tttaatagtggactcttgttccaaactggaacaacactcaactctatctcgggctattct tttgatttataaggg attttgccgatttcggtctattggttaaaaaatgagctgatttaacaaaaatttaacgcg aattttaacaaaata ttaacgtttacaattttatggtgcactctcagtacaatctgctctgatgccgcatagtta agccagccccgacac ccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacaga caagctgtgaccgtc tccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaag ggcctcgtgatacgc ctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcactttt cggggaaatgtgcgc ggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaa taaccctgataaatg cttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttatt cccttttttgcggca ttttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagat cagttgggtgcacga gtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaa gaacgttttccaatg atgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaa gagcaactcggtcgc cgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatctt acggatggcatgaca gtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttactt ctgacaacgatcgga ggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgat cgttgggaaccggag ctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaaca acgttgcgcaaacta ttaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcg gataaagttgcagga ccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggt gagcgtggaagccgc ggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacg acggggagtcaggca actatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattgg taactgtcagaccaa gtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctag gtgaagatccttttt gataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagacccc gtagaaaagatcaaa ggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaacca ccgctaccagcggtg gtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcaga gcgcagataccaaat actgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcct acatacctcgctctg ctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggac tcaagacgatagtta ccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggag cgaacgacctacacc gaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaag gcggacaggtatccg gtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctgg tatctttatagtcct gtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcgg agcctatggaaaaac gccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgt

Some of the embodiments, advantages, features, and uses of the technology disclosed herein will be more fully understood from the Examples below. The Examples are intended to illustrate some of the benefits of the present disclosure and to describe particular embodiments but are not intended to exemplify the full scope of the disclosure and, accordingly, do not limit the scope of the disclosure. EXAMPLES

Example 1. Generation of genetically engineered hematopoietic cells using fusion polypeptides, including base editing fusion polypeptides

This example demonstrates generation of fusion polypeptides and their use in generating genetically engineered cells, such as genetically engineered hematopoietic cells. Casl2a/Cpfl gRNAs are synthesized using gRNA target domains directed to target sequences of interest.

Peripheral blood mononuclear cells are collected from healthy donor subject by apheresis following hematopoietic stem cell mobilization. Alternatively, frozen CD34+ HSCs derived from mobilized peripheral blood (mPB) are purchased, for example, from Hemacare or Fred Hutchinson Cancer Center and thawed according to manufacturer’s instructions. -IxlO ⁶ HSCs are thawed and cultured in StemSpan SFEM medium supplemented with StemSpan CC110 cocktail (StemCell Technologies) for 24-48 h before electroporation with RNP.

The donor or purchased CD34+ cells are electroporated with the fusion polypeptide and gRNAs targeting a targeting sequence of interest. To electroporate HSCs, 1.5 xlO ⁵ cells are pelleted and resuspended in 20 pL Lonza P3 solution and mixed with 10 pL RNP comprising the fusion polypeptides and gRNA. CD34+ HSCs are electroporated using the Lonza Nucleofector 2 and the Human P3 Cell Nucleofection Kit (VPA-1002, Lonza).

The edited cells are cultured for less than 48 hours. Upon harvest, the cells are washed, resuspended in the final formulation, and cryopreserved.

A representative sample of the edited HSCs (e.g., a portion of or all cells of the time point aliquots) is evaluated for viability, editing efficiency at the target sequence, and/or expression of exemplary target region genes, or absence thereof, by staining using targetspecific antibody and analyzed by flow cytometry. Edited HSC populations may also be assessed for development and differentiation into particular cell types, such as macrophages, T cells, B cells, and myeloid cells.

Genomic DNA analysis

For all genomic analysis, DNA is harvested from cells, amplified with primers flanking the target region, purified and the allele modification frequencies are analyzed using appropriate methods known in the art. Analyses are performed using a reference sequence from a mock-transfected sample.

Flow cytometry analysis

The gRNA-edited cells may also be evaluated for surface expression of target gene encoded protein, for example by flow cytometry analysis (FACS). Live CD34+ HSCs are stained for target gene protein using a target- specific antibody and analyzed by flow cytometry on the Attune NxT flow cytometer (Life Technologies).

Viability of edited cells

At O, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, and/or 48 hours post-ex vivo editing (e.g., 4, 24, and 48 hours post-ex vivo editing), the percentages of viable, edited cells, and control cells are quantified using flow cytometry and the 7AAD viability dye. Cells edited using the exemplary gRNAs or sgRNAs described herein may be viable and remain viable over time following electroporation and gene editing. This is similar to what is observed in the control mock edited cells.

Example 2: Assessing genetic editing in vitro

To assess the ability of fusion polypeptides described herein to effect targeted DNA modifications in cultured cells, fusion polypeptides, such as any of the fusion polypeptides described herein (e.g., such as those shown in the plasmids shown in FIGs. 1-12 or provided by any of SEQ ID NOs: 24-31, and gRNAs targeting a desired genetic locus are introduced into appropriate cultured cell populations, such as a cell line or cells sourced from subject, including healthy subjects or patient populations of interest. In some experiments, expression plasmids encoding the fusion protein and gRNAs, such as the plasmids shown in FIGs. 1-12, are generated and used to produce the fusion polypeptides.

The fusion polypeptides may be incubated with gRNAs to form a ribonucleoprotein (RNP) complex, and then used to transfect host cells.

After sufficient time, 48, 72, or 120 hours after electroporation, genomic DNA is extracted from edited cells and from control (non-edited) cells. Sequencing, such as Sanger sequencing of a target sequence, whole genome sequencing, may be performed to assess the efficiency of genomic modification and determine any off-target editing. Example 3: Treating a subject with edited cells

An example treatment regimen using the methods, cells, and agents described herein for acute myeloid leukemia is provided below.

1) Identify a patient with AML who is a candidate for receiving a hematopoietic cell transplant (HCT);

2) Identify a HCT donor with matched HLA haplotypes, using standard methods and techniques;

3) Extract the bone marrow from the donor;

4) Genetically manipulate the donor bone marrow cells ex vivo. Briefly, introduce targeted modifications (deletion, substitution) of a lineage- specific cell-surface antigen using a gRNA and any of the fusion polypeptides described herein. Cells may be evaluated for characteristics to determine their ability to differentiate and the ability to engraft the patient and mediate graft-vs-tumor (GVT) effects.

Optional Steps 5-7:

In some embodiments, Steps 5-7 provided below may be performed (once or multiple times) in an exemplary treatment method as described herein:

5) Pre-condition the AML patient using standard techniques, such as infusion of chemotherapy agents (e.g., etoposide, cyclophosphamide) and/or irradiation;

6) Administer the engineered donor bone marrow to the AML patient, allowing for successful engraftment;

7) Follow up with a cytotoxic agent, such as immune cells expressing a chimeric receptor (e.g., CAR T cell) or antibody-drug conjugate, wherein the epitope to which the cytotoxic agent binds is the same epitope that was modified and is no longer present on the engineered cells. The targeted therapy should thus specifically target the antigen, without simultaneously eliminating the bone marrow graft, in which the epitope is not present.

Optional Steps 8-10:

In some embodiments, Steps 8-10 may be performed (once or multiple times) in an exemplary treatment method as described herein:

8) Administer a cytotoxic agent, such as immune cells expressing a chimeric receptor (e.g., CAR T cell) or antibody-drug conjugate that targets an epitope of an antigen. This targeted therapy would be expected to eliminate both cancerous cells as well as the patient’s non-cancerous cells; 9) Pre-condition the AML patient using standard techniques, such as infusion of chemotherapy agents;

10) Administer the engineered donor bone marrow to the AML patient, allowing for successful engraftment.

The steps 8-10 result in the elimination of the patient’s cancerous and normal cells expressing the targeted protein, while replenishing the normal cell population with donor cells that are resistant to the targeted therapy.

REFERENCES

All publications, patents, patent applications, publication, and database entries (e.g., sequence database entries) mentioned herein, e.g., in the Background, Summary, Detailed Description, Examples, and/or References sections, are hereby incorporated by reference in their entirety as if each individual publication, patent, patent application, publication, and database entry was specifically and individually incorporated herein by reference. In case of conflict, the present application, including any definitions herein, will control.

EQUIVALENTS AND SCOPE

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the embodiments described herein. The scope of the present disclosure is not intended to be limited to the above description, but rather is as set forth in the appended claims.

Articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between two or more members of a group are considered satisfied if one, more than one, or all of the group members are present, unless indicated to the contrary or otherwise evident from the context. The disclosure of a group that includes “or” between two or more group members provides embodiments in which exactly one member of the group is present, embodiments in which more than one members of the group are present, and embodiments in which all of the group members are present. For purposes of brevity those embodiments have not been individually spelled out herein, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.

It is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitation, element, clause, or descriptive term, from one or more of the claims or from one or more relevant portion of the description, is introduced into another claim. For example, a claim that is dependent on another claim can be modified to include one or more of the limitations found in any other claim that is dependent on the same base claim. Furthermore, where the claims recite a composition, it is to be understood that methods of making or using the composition according to any of the methods of making or using disclosed herein or according to methods known in the art, if any, are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

Where elements are presented as lists, e.g., in Markush group format, it is to be understood that every possible subgroup of the elements is also disclosed, and that any element or subgroup of elements can be removed from the group. It is also noted that the term “comprising” is intended to be open and permits the inclusion of additional elements or steps. It should be understood that, in general, where an embodiment, product, or method is referred to as comprising particular elements, features, or steps, embodiments, products, or methods that consist, or consist essentially of, such elements, features, or steps, are provided as well. For purposes of brevity those embodiments have not been individually spelled out herein, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.

Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in some embodiments, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. For purposes of brevity, the values in each range have not been individually spelled out herein, but it will be understood that each of these values is provided herein and may be specifically claimed or disclaimed. It is also to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.

In addition, it is to be understood that any particular embodiment of the present invention may be explicitly excluded from any one or more of the claims. Where ranges are given, any value within the range may explicitly be excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and/or methods described herein, can be excluded from any one or more claims. For purposes of brevity, all of the embodiments in which one or more elements, features, purposes, or aspects is excluded are not set forth explicitly herein.