STATEMENT REGARDING FEDERAL FUNDING

This invention was made with government support under OD021369 awarded by the National Institutes of Health. The government has certain rights in the invention.

TECHNICAL FIELD

This document relates to compositions, methods, and systems for site-specific integration (e.g., stable integration) of a nucleic acid (e.g., large nucleic acid) into the genome of a cell (e.g., a prokaryotic cell or a eukaryotic cell such as a plant cell or an animal cell). For example, this document provides compositions, methods, and systems for stably integrating one or more nucleic acids into a target site within the genome of a cell that include (a) a genome-editing system having (i) a polypeptide having a DNA binding domain and, optionally, a polymerase and (ii) a nucleic acid molecule including a guide sequence that is complementary to the target site and a nucleic acid sequence that encodes an acceptor attachment (attA) site, (b) a donor nucleic acid molecule including a nucleic acid cargo and a donor attachment (attD) site, and (c) an integrase (e.g., a large serine recombinase (LSR)) that can target the attA site and the attD site, where the integrase can facilitate recombination between the attA site and the attD site.

BACKGROUND INFORMATION

Current gene integration approaches rely on DNA double-stranded breaks (DSBs) to direct cellular DNA repair pathways such as homologous recombination (HR). These approaches generally suffer from low insertion efficiency, high indel rates, and cargo size limitations. Additional gene integration approaches such as transposase-mediated integration and lentiviral-mediated integration are not site-specific, and can result in variable gene expression, silenced gene expression, insertional mutagenesis, and/or other undesired events

Despite the recent advances in genome engineering technologies, there remains a need for an efficient method to stably and site-specifically integrate multi-kilobase DNA cargos into human and other eukaryotic cell genomes. SUMMARY

This document provides compositions, methods, and systems for integrating (e.g., stably integrating) nucleic acid (e.g., large nucleic acid) into the genome of a cell (e.g., prokaryotic cell or a eukaryotic cell such as a plant cell or an animal cell). For example, this document provides compositions, methods, and systems for stably integrating one or more nucleic acids into a target site within the genome of a cell that include (a) a genome- editing system having (i) a polypeptide having a DNA binding domain and, optionally, a polymerase and (ii) a nucleic acid molecule including a guide sequence that is complementary to the target site and a nucleic acid sequence that encodes an attA site, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site. For example, when a genome-editing system provided herein is administered to a cell, the genome-editing system can insert the attA into the genome at the target site, and the integrase can facilitate recombination between the attA site and the attD site thereby integrating the donor nucleic acid molecule into the genome.

As demonstrated herein, a genome-editing system (e.g., a prime-editor system) can be used together with an integrase (e.g., a LSR) to stably integrate multi-kilobase DNA cargos into human and other eukaryotic cell genomes. The compositions, methods, and systems provided herein not only provide precise control over the genomic integration site (thus reducing or eliminating the risk of insertional mutagenesis), but can allow the site-specific integration of large (e.g., multi-kilobase) nucleic acid cargos into the genome. The compositions, methods, and systems provided herein can be applied to any appropriate gene editing application including, without limitation, gene therapy methods, gene transfer methods, production of transgenic plants, production of gene knock-out plants, and production of gene knock-out non-human animal models.

In general, one aspect of this document features systems for stably integrating one or more nucleic acid sequences into a genome of a cell. The systems can include, or consist essentially of, administering to a cell: (a) a genome-editing system that can insert an attA sequence into a target site within a genome of the cell; (b) a donor nucleic acid molecule comprising a nucleic acid cargo and a attD sequence; and (c) an integrase that targets the attA sequence and the attD site and can facilitate recombination between the attA site and the attD site. The cell can be a mammalian cell (e.g., a human cell). The cell can be a plant cell. The cell can be a prokaryotic cell. The genome-editing system can include (i) a polypeptide comprising a DNA binding domain and (ii) a nucleic acid comprising a guide sequence that is complementary to the target site within the genome and a sequence that encodes the attA sequence. The DNA binding domain can be present in polypeptide selected from a Cas9 polypeptide, a Cas12 polypeptide, a zinc finger polypeptide, and a transcription activator-like effector (TALE) polypeptide. The polypeptide including the DNA binding domain can be a polymerase. The polymerase can be a reverse transcriptase (RT) selected from the group consisting of a Moloney murine leukemia virus (M-MLV) RT, an avian myeloblastosis virus (AMV) RT, and a human immunodeficiency virus type 1 (HIV-1) RT. The attA sequence can include from about 20 to about 100 nucleic acids. The attA sequence can comprise, consist essentially of, or consist of any one of SEQ ID NOs: 11-84 and SEQ ID NO:254. The attD sequence can include from about 20 to about 100 nucleic acids. The attD sequence can comprise, consist essentially of, or consist of any one of SEQ ID NOs: 159-232. The integrase can be a LSR. The LSR can have an amino acid sequence containing a motif set forth in any one of SEQ ID NOs:233-245. The LSR can have an amino acid sequence having at least 70% sequence identity to the sequence of any one of SEQ ID NOs: 85-158. The LSR can have an amino acid sequence having at least 90% sequence identity to the sequence of any one of SEQ ID NOs: 85-158. The LSR can comprise, consist essentially of, or consist of an amino acid sequence set forth in any one of SEQ ID NOs: 85-158. The donor nucleic acid molecule can be from about 250 nt to about 30 kb.

In another aspect, this document features methods for stably integrating one or more nucleic acid sequences into a genome of a cell. The methods can include, or consist essentially of, administering to a cell: (a) a genome-editing system that can insert an attA sequence into a target site within a genome of the cell; (b) a donor nucleic acid molecule comprising a nucleic acid cargo and an attD sequence; and (c) an integrase that targets the attA sequence and the attD site; where the genome- editing system integrates the attA sequence into the target site, and where the integrase facilitates recombination between the attA sequence and the attD sequence thereby integrating the donor nucleic acid molecule into the genome of the cell. The cell can be a T cell, a natural killer (NK) cell, a non-human embryonic stem cell, an induced pluripotent stem cell (iPSC), a hematopoietic stem cell (HSC), a liver cell, a muscle cell, a monocytes, a B cell, a neuron, an astrocyte, or a microglial cell. The cell can be a T cell and the nucleic acid sequence can encode a chimeric antigen receptor polypeptide or an engineered T cell receptor. The cell is a NK cell and the nucleic acid sequence can encode a T cell receptor or an engineered natural killer cell receptor. The cell can be a mammalian cell (e.g., a human cell). The cell can be a plant cell. The genome- editing system can include (i) a polypeptide comprising a DNA binding domain and (ii) a nucleic acid comprising a guide sequence that is complementary to the target site within the genome and a sequence that encodes the attA sequence. The DNA binding domain can be present in a polypeptide selected from a Cas9 polypeptide, a Cas12 polypeptide, a zinc finger polypeptide, and a TALE polypeptide. The polypeptide comprising the DNA binding domain can be a polymerase. The polymerase can be an RT selected from the group consisting of a M-MLV RT, an AMV RT, and a HIV-1 RT. The attA sequence can comprise, consist essentially of, or consist of any one of SEQ ID NOs: 11-84 and SEQ ID NO:254. The attD sequence can comprise, consist essentially of, or consist of any one of SEQ ID NOs: 159-232. The integrase can be a LSR. The LSR can have an amino acid sequence containing a motif set forth in any one of SEQ ID NOs: 233 -245. The LSR can have an amino acid sequence having at least 70% sequence identity to the sequence of any one of SEQ ID NOs:85-l 58. The LSR can have an amino acid sequence having at least 90% sequence identity to the sequence of any one of SEQ ID NOs: 85- 158. The LSR can comprise, consist essentially of, or consist of an amino acid sequence set forth in any one of SEQ ID NOs: 85- 158.

In another aspect, this document features methods for labelling a polypeptide encoded by an endogenous nucleic acid within a cell. The methods can include, or consist essentially of, administering to a cell: (a) a genome-editing system that can insert an attA sequence into a target site within a genome of the cell; (b) a donor nucleic acid molecule comprising a nucleic acid cargo encoding a detectable label and an attD sequence; and (c) an integrase that targets the attA sequence and the attD site; where the genome-editing system integrates the attA sequence into the target site, and where the integrase facilitates recombination between the attA sequence and the attD sequence thereby integrating the donor nucleic acid molecule into the genome of the cell such that the cell expresses a fusion polypeptide including the polypeptide encoded by the endogenous nucleic acid fused to the detectable label. The detectable label can be a HiBiT tag, a HaloTag, a Flag tag, a HA tag, a MS2/PP7 tag, a Sun/Moon tag, a poly(His) tag, a mCherry polypeptide, a green fluorescent polypeptide (GFP), a glutathione-S-transferase (GST), a luciferase, a horseradish peroxidase (HRP), an alkaline phosphatase (AP), or a apurinic/apyrimidinic endodeoxyribonuclease 2 (APEX2) polypeptide. The cell can be a mammalian cell (e.g., a human cell). The cell can be a plant cell. The genome-editing system can include (i) a polypeptide comprising a DNA binding domain and (ii) a nucleic acid comprising a guide sequence that is complementary to the target site within the genome and a sequence that encodes the attA sequence. The DNA binding domain can be present in a polypeptide selected from a Cas9 polypeptide, a Cas12 polypeptide, a zinc finger polypeptide, and a TALE polypeptide. The polypeptide including the DNA binding domain can be a polymerase. The polymerase can be a RT selected from the group consisting of a M-MLV RT, an AMV RT, and a HIV-1 RT. The attA sequence can comprise, consist essentially of, or consist of any one of SEQ ID NOs: 11-84 and SEQ ID NO:254. The attD sequence can comprise, consist essentially of, or consist of any one of SEQ ID NOs: 159-232. The integrase can be a LSR. The LSR can have an amino acid sequence containing a motif set forth in any one of SEQ ID NOs:233-245. The LSR can have an amino acid sequence having at least 70% sequence identity to the sequence of any one of SEQ ID NOs: 85- 158. The LSR can have an amino acid sequence having at least 90% sequence identity to the sequence of any one of SEQ ID NOs: 85-158. The LSR can comprise, consist essentially of, or consist of an amino acid sequence set forth in any one of SEQ ID NOs: 85-158.

In another aspect, this document features methods for making a non-human transgenic organism. The methods can include, or consist essentially of, administering to an embryonic stem cell of a non-human organism: (a) a genome-editing system that can insert an attA sequence into a target site within a genome of the embryonic stem cell; (b) a donor nucleic acid molecule comprising a transgene and an attD sequence; and (c) an integrase that targets the attA sequence and the attD site; where the genome-editing system integrates the attA sequence into the target site, and where the integrase facilitates recombination between the attA sequence and the attD sequence thereby integrating the donor nucleic acid molecule into the genome of the cell such that the cell expresses the transgene. The cell can be a non- human mammalian cell. The cell can be a plant cell. The transgene expressed by the plant cell can be a herbicide resistance polypeptide. The genome-editing system can include (i) a polypeptide comprising a DNA binding domain and (ii) a nucleic acid comprising a guide sequence that is complementary to the target site within the genome and a sequence that encodes the attA sequence. The DNA binding domain can be present in a polypeptide selected from a Cas9 polypeptide, a Cas12 polypeptide, a zinc finger polypeptide, and a TALE polypeptide. The polypeptide including the DNA binding domain can be a polymerase. The polymerase can be an RT selected from the group consisting of a M-MLV RT, an AMV RT, and a HIV-1 RT. The attA sequence can comprise, consist essentially of, or consist of any one of SEQ ID NOs: 11-84 and SEQ ID NO:254. The attD sequence can comprise, consist essentially of, or consist of any one of SEQ ID NOs: 159-232. The integrase can be a LSR. The LSR can have an amino acid sequence containing a motif set forth in any one of SEQ ID NOs:233-245. The LSR can have an amino acid sequence having at least 70% sequence identity to the sequence of any one of SEQ ID NOs: 85-158. The LSR can have an amino acid sequence having at least 90% sequence identity to the sequence of any one of SEQ ID NOs: 85-158. The LSR can comprise, consist essentially of, or consist of an amino acid sequence set forth in any one of SEQ ID NOs: 83-158.

In another aspect, this document features methods for making a non-human organism having reduced or eliminated levels of a polypeptide. The methods can include, or consist essentially of, administering to an embryonic cell of a non-human organism: (a) a genome- editing system that can insert an attA sequence into a target site within a genome of the cell;

(b) a donor nucleic acid molecule comprising a nucleic acid cargo and an attD sequence; and

(c) an integrase that targets the attA sequence and the attD site; where the genome-editing system integrates the attA sequence into the target site, and where the integrase facilitates recombination between the attA sequence and the attD sequence thereby integrating the donor nucleic acid molecule into the genome of the cell such that the endogenous nucleic acid sequence encoding the polypeptide is interrupted and expression of the polypeptide is reduced or eliminated. The nucleic acid cargo can include a stop codon. The nucleic acid cargo can include a nucleic acid encoding a selectable marker. The nucleic acid cargo can include nucleic acid encoding a detectable label. The cell can be a non-human mammalian cell. The cell can be a plant cell. The genome-editing system can include (i) a polypeptide comprising a DNA binding domain and (ii) a nucleic acid comprising a guide sequence that is complementary to the target site within the genome and a sequence that encodes the attA sequence. The DNA binding domain can be present in a polypeptide selected from a Cas9 polypeptide, a Cas12 polypeptide, a zinc finger polypeptide, and a TALE polypeptide. The polypeptide including the DNA binding domain can be a polymerase. The polymerase can be an RT selected from the group consisting of a M-MLV RT, an AMV RT, and a HIV-1 RT. The attA sequence can comprise, consist essentially of, or consist of any one of SEQ ID NOs: 11-84 and SEQ ID NO:254. The attD sequence can comprise, consist essentially of, or consist of any one of SEQ ID NOs: 159-232. The integrase can be a LSR. The LSR can have an amino acid sequence containing a motif set forth in any one of SEQ ID NOs:233- 245. The LSR can have an amino acid sequence having at least 70% sequence identity to the sequence of any one of SEQ ID NOs: 85-158. The LSR can have an amino acid sequence having at least 90% sequence identity to the sequence of any one of SEQ ID NOs: 85-158. The LSR can comprise, consist essentially of, or consist of an amino acid sequence set forth in any one of SEQ ID NOs:85-158.

In another aspect, this document features methods for treating a mammal having a disease or disorder. The methods can include, or consist essentially of, administering to a mammal having a disease or disorder: (a) a genome-editing system that can insert an attA sequence into a target site within a genome of a cell within the mammal; (b) a donor nucleic acid molecule comprising a nucleic acid cargo encoding a therapeutic gene product and a attD sequence; and (c) an integrase that targets the attA sequence and the attD site; where the genome-editing system integrates the attA sequence into the target site, and where the integrase facilitates recombination between the attA sequence and the attD sequence thereby integrating the donor nucleic acid molecule into the genome of the cell such that the cell produces the therapeutic gene product. The therapeutic polypeptide can be an adenosine deaminase polypeptide, an α-1 antitrypsin polypeptide, a cystic fibrosis transmembrane conductance regulator (CFTR) polypeptide, a β-hemoglobin (HBB) polypeptide, an oculocutaneous albinism II (OCA2) polypeptide, a Huntingtin (HTT) polypeptide, a dystrophia myotonica-protein kinase (DMPK) polypeptide, a low-density lipoprotein receptor (LDLR) polypeptide, an apolipoprotein B (APOB) polypeptide, a neurofibromin 1 (NF1) polypeptide, a polycystic kidney disease 1 (PKD1) polypeptide, a polycystic kidney disease 2 (PKD2) polypeptide, a coagulation factor VIII (F8) polypeptide, a dystrophin (DMD) polypeptide, a phosphate-regulating endopeptidase homologue X-linked (PHEX) polypeptide, a methyl-CpG-binding protein 2 (MECP2) polypeptide, a ubiquitin-specific peptidase 9Y, Y-linked (USP9Y) polypeptide, a carbamoyl-phosphate synthase 1 (CPS1) polypeptide, an ATP binding cassette subfamily A member 4 (ABCA4) polypeptide, an fatty acid elongase 4 (ELOVL) polypeptide, amyosin VIIA (MY07A) polypeptide, an usher syndrome 1C (USH1C) polypeptide, a cadherin related 23 (CDH23) polypeptide, a protocadherin related 15 (PCDH15) polypeptide, an usher syndrome 1G (USH1G) polypeptide, an usher syndrome 2A (USH2A) polypeptide, an adhesion G protein- coupled receptor VI (ADGRV1) polypeptide, a whirlin (WHRN) polypeptide, a clarin 1 (CLRN1) polypeptide, a retinitis pigmentosa 1 (RP1) polypeptide, an eyes shut homolog (EYS) polypeptide, a lipoprotein (a) (LPA) polypeptide, a lipoprotein lipase (LPL) polypeptide, an apolipoprotein C2 (AP0C2) polypeptide, an apolipoprotein A5 (AP0A5) polypeptide, a lipase maturation factor 1 (LMF1) polypeptide, a glycosylphosphatidylinositol anchored high density lipoprotein binding protein 1 (GPIHBP1) polypeptide, a proprotein convertase subtilisin/kexin type 9 (PCSK9) polypeptide, a ryanodine receptor 2 (RYR2) polypeptide, a calsequestrin 2 (CASQ2) polypeptide, a myosin heavy chain 7 (MYH7) polypeptide, a myosin binding protein C3 (MYBPC3) polypeptide, a troponin T2, cardiac type (TNNT2) polypeptide, and a troponin 13, cardiac type (TNNI3) polypeptide, or a C9orf72 polypeptide. The mammal can be a human. The genome-editing system can include (i) a polypeptide comprising a DNA binding domain and (ii) a nucleic acid comprising a guide sequence that is complementary to the target site within the genome and a sequence that encodes the attA sequence. The DNA binding domain can be present in a polypeptide selected from a Cas9 polypeptide, a Cas12 polypeptide, a zinc finger polypeptide, and a TALE polypeptide. The polypeptide including the DNA binding domain can be a polymerase. The polymerase can be an RT selected from the group consisting of a M-MLV RT, an AMV RT, and a HIV-1 RT. The attA sequence can comprise, consist essentially of, or consist of any one of SEQ ID NOs: 11-84 and SEQ ID NO:254. The attD sequence can comprise, consist essentially of, or consist of any one of SEQ ID NOs: 159-232. The integrase can be a LSR. The LSR can have an amino acid sequence containing a motif set forth in any one of SEQ ID NOs:233- 245. The LSR can have of an amino acid sequence having at least 70% sequence identity to the sequence of any one of SEQ ID NOs: 85- 158. The LSR can have an amino acid sequence having at least 90% sequence identity to the sequence of any one of SEQ ID NOs: 85-158. The LSR can comprise, consist essentially of, or consist of an amino acid sequence set forth in any one of SEQ ID NOs:85-158.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

Figures 1A - 1C. Schematic images of mechanism for using a prime editor in combination with a LSR for programmable recombination of multiple kilobase cargo into the genome. Figure 1 A contains a schematic for using prime editing with a LSR supplied independently (e.g., in trans). Figure IB contains a schematic for using prime editing with integrase supplied fused to a component of a prime editor complex (e.g., in cis). Figure 1C contains a schematic image showing guided delivery of the prime editor to a nucleic acid target site using pegRNA & ngRNA (left) or using two twinPE pegRNAs (right). Figures 2A - 2B. Schematic images of exemplary methods for using a prime editor in combination and a LSR in trans for programmable recombination of multiple kilobase cargo into the genome. Figure 2A contains a schematic of an exemplary method for a one- step transfection to deliver a prime editing system and a LSR to cells. Figure 2B contains a schematic of an exemplary method for a two-step transfection to deliver a prime editing system and a LSR to cells.

Figure 3. Sequencing results demonstrating that prime editing can be used for targeted insertion of an attA site. Sequencing results of Bxbl are, from top to bottom, SEQ ID NOs:246 to 249. Sequencing results of PaOl are, from top to bottom, SEQ ID NOs:250 and 251.

Figure 4. PCR validation of donor integration at an attA site.

Figures 5A - 5B. Sequencing results demonstrating site-specific donor integration. Figure 5A contains results using a Bxbl LSR (SEQ ID NO:252). Figure 5B contains results using a PaOl LSR (SEQ ID NO:253).

Figure 6. Evaluation of attA length. Truncations of an exemplary minimal attB site (SEQ ID NO:254) are shown.

Figure 7. qPCR analysis showing donor integration using 1 pegRNA.

Figures 8A - 8B. ddPCR analysis showing donor integration. Figure 8A. Donor integration at the LMNB1 locus using 1 pegRNA. Figure 8B. Donor integration at the ACTB locus using 1 pegRNA.

Figure 9. qPCR analysis showing donor integration using 2 pegRNAs at the AAVS1 locus.

Figure 10. ddPCR analysis showing donor integration at the AAVS1 locus using 2 pegRNAs and LSR delivery in trans.

DETAILED DESCRIPTION

This document provides compositions, methods, and systems for integrating (e.g., stably integrating) nucleic acid (e.g., large nucleic acid) into the genome of a cell (e.g., a prokaryotic cell or a eukaryotic cell such as a plant cell or an animal cell). For example, this document provides systems for stably integrating one or more nucleic acids into a target site within the genome of a cell that include (a) a genome- editing system having (i) a polypeptide having a DNA binding domain and, optionally, a polymerase and (ii) a nucleic acid molecule including a guide sequence that is complementary to the target site and a nucleic acid sequence that encodes an attA site, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site. For example, when a genome-editing system provided herein is administered to a cell, the genome- editing system can insert the attA into the genome at the target site, and the integrase can facilitate recombination between the attA site and the attD site thereby integrating the donor nucleic acid molecule into the genome.

The compositions, methods, and systems provided herein (e.g., a system for stably integrating one or more nucleic acids into a target site within the genome of a cell including (a) a genome- editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site) can be used to integrate (e.g., stably integrate) a nucleic acid into a genomes of any appropriate type of cell. In some cases, the compositions, methods, and systems provided herein can be used to integrate nucleic acid (e.g., large nucleic acid) into a prokaryotic cell. In some cases, the compositions, methods, and systems provided herein can be used to integrate nucleic acid (e.g., large nucleic acid) into a eukaryotic cell. Examples of cell types that can have a nucleic acid stably integrated within the genome as described herein include, without limitation, stem cells (e.g., non-human embryonic stem cells, induced pluripotent stem cells (iPSCs), and hematopoietic stem cells (HSCs)), immune cells (e.g., T cells, macrophages, monocytes, B cells, and natural killer (NK) cells), liver cells, muscle cells, and brain cells (e.g., neurons, astrocytes, and microglia). For example, a system including (a) a genome-editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site) can be used to integrate (e.g., stably integrate) a nucleic acid into a plant cell or a mammalian cell. Examples of plants whose cells can have a nucleic acid stably integrated into a target site within the genome as described herein include, without limitation, wheat, corn, soy, rice, tobacco, Arabidopsis thaliana, cacao, banana, and sunflower. Examples of mammals whose cells can have a nucleic acid stably integrated into a target site within the genome as described herein include, without limitation, humans, non- human primates such as chimpanzees and monkeys, dogs, cats, horses, cows, pigs, sheep, mice, rats, rabbits, guinea pigs, birds, fish (e.g., zebrafish (Danio rerio), medaka (Oryzias talipes), and turquoise killifish (Nothobranchius furzeri)), nematodes (e.g., Caenorhabditis elegans), and flies (e.g., Drosophila melanogaster).

A genome-editing system in a system for stably integrating one or more nucleic acids into a target site within the genome of a cell provided herein can include (i) a polypeptide having a DNA binding domain and, optionally, a polymerase and (ii) a nucleic acid molecule including a guide sequence that is complementary to the target site and a nucleic acid sequence that encodes an attA site. A polypeptide having a DNA binding domain and, optionally, a polymerase can include any appropriate DNA binding domain. In some cases, a DNA binding domain can be included in a polypeptide including a DNA binding domain. For example, a DNA binding domain can be included in a polypeptide including a DNA binding domain and including nuclease activity. For example, a DNA binding domain can be included in a polypeptide including a DNA binding domain and including nickase activity.

A DNA binding domain can be included in any appropriate polypeptide having nuclease activity. Examples of nucleases include, without limitation, clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) polypeptides, zinc-finger nucleases (ZFNs), and transcription activator- like effector (TALE) polypeptides. In some cases, a nuclease can be as described elsewhere (see, e.g., Urnov and Rebar, Biochem. Pharmacol., 64(5-6): 919-23 (2002); and Miller et al., Nat. Biotechnol., 29(2): 143-8 (2011)).

In some cases, a DNA binding domain can be included a Cas polypeptide. A Cas polypeptide can be any appropriate Cas polypeptide. In some cases, a Cas polypeptide can be isolated from an organism (e.g., a bacterium). In some cases, a Cas polypeptide can be a recombinant polypeptide. In some cases, a Cas polypeptide can be a synthetic polypeptide. Examples of Cas polypeptides include, without limitation, Cas9 polypeptides (e.g., a Cas9 nuclease or a Cas9 nickase) such as Cas9 polypeptides from Streptococcus pyogenes (SpCas9 polypeptides) and Cas9 polypeptides from Staphylococcus aureus (SaCas9 polypeptides), Cas12 polypeptides (e.g., a Cas12 nuclease or a Cas12 nickase). A Cas polypeptide having a DNA binding domain can have any appropriate amino acid sequence. Examples of Cas polypeptide sequences include, without limitation, amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6. In some cases, a Cas polypeptide having a DNA binding domain can have one or more amino acid modifications (e.g., one or more insertions, one or more deletions, and/or one or more substitutions) relative to a Cas polypeptide described herein (e.g., SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 5, and SEQ ID NO:6), provided the Cas polypeptide maintains the ability to cleave nucleic acid (e.g., maintains its nuclease activity and/or its nickase activity). In some cases, a Cas polypeptide having a DNA binding domain can have at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, provided the Cas polypeptide maintains the ability to cleave nucleic acid (e.g., maintains its nuclease activity and/or its nickase activity).

In some cases, a Cas polypeptide having a DNA binding domain can include one or more additional polypeptides (e.g., a subcellular localization signal such as a nuclear localization signal (NLS)).

In some cases, a Cas polypeptide having a DNA binding domain can be as described elsewhere (see, e.g., Cong et al., Science 339(6121):819-23 (2013); Hsu et al., Nat. Biotechnol., 31:827-832 (2013); Jinek et al., Science, 337(6096): 816-21 (2012); Mali et al., Science, 339(6121):823-6 (2013); Nishimasu et al., Cell, 156(5):935-49 (2014); and Friedland et al., Genome Biol., 16:257 (2015)).

In cases where a polypeptide having a DNA binding domain includes a polymerase, the polymerase can be any appropriate polymerase. In some cases, the polymerase can be a transcriptase (e.g., reverse transcriptase). Examples of polymerases include, without limitation, reverse transcriptases from a Moloney murine leukemia virus (M-MLV RTs), reverse transcriptases from an avian myeloblastosis virus (AMV RTs), and reverse transcriptases from a human immunodeficiency virus type 1 (HIV-1 RTs). In some cases, a polymerase can be as described elsewhere (see, e.g., Gao et al., bioRxiv doi.org/10.1101/2021.11.05.467423 (2021)). A polymerase (e.g., a reverse transcriptase) can have any appropriate amino acid sequence. Examples of polymerase sequences include, without limitation, amino acid sequences set forth in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10. In some cases, a polymerase can have one or more amino acid modifications (e.g., one or more insertions, one or more deletions, and/or one or more substitutions) relative to a polymerase described herein (e.g, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10), provided the polymerase maintains the ability to synthesize nucleic acid (e.g, maintains its polymerase activity). In some cases, a polymerase can have at least 70% (e.g, 70%, 75%, 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, or 99%) sequence identity to any one of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, provided the polymerase maintains the ability to synthesize nucleic acid (e.g, maintains its polymerase activity).

In some cases, a polymerase (e.g, a reverse transcriptase) can include one or more additional polypeptides (e.g, a subcellular localization signal such as a NLS).

In some cases, a polymerase (e.g, a reverse transcriptase) can be as described elsewhere (see, e.g, Baranauskas et al. Protein Eng. Des. Sei., 25(10):657-68 (2012); Anzalone et al. Nature, 576(7785): 149-157 (2019); loannidi et al, BioRxiv, DOI 10.1101/2021.11.01.466786 (2021); Perbal et al, Retrovirology, 5:49 (2008); Komshi et al, Biotechnol. Lett., 34(7): 1209-15 (2012); Hu et al. Cold Spring Harb. Perspect. Med, 2(10):a006882 (2012); UniProt Accession No. Q9WJQ2; and Japanese Patent Application Publication JP2012120506A).

A nucleic acid molecule including a guide sequence that is complementary to a target site and a nucleic acid sequence that encodes an attA site in a genome editing system provided herein can include any appropriate guide sequence. In some cases, a guide sequence can be a guide RNA (gRNA). A guide sequence can be complementary to (e.g, can be designed to be complementary to) any appropriate target site. It will be appreciated that a target site within a genome can be designed specifically for the desired outcome of the stably integrated nucleic acid. For example, when a stably integrated nucleic acid is designed to express a transgene, the target site can be designed such that expression of any endogenous nucleic acid is not disrupted. For example, when a stably integrated nucleic acid is designed to disrupt and/or replace an endogenous nucleic acid encoding a polypeptide, the target site can be designed to be within the endogenous nucleic acid encoding the polypeptide (e.g., a coding sequence within that endogenous nucleic acid or a non-coding sequence within that endogenous nucleic acid).

A nucleic acid molecule including a guide sequence that is complementary to a target site and a nucleic acid sequence that encodes an attA site in a genome editing system provided herein can include any appropriate nucleic acid sequence that encodes an attA site. An attA site, as used herein, is an attachment site for an integrase described herein. In some cases, an attA site can be an acceptor attachment site derived from a bacterial target sequence (e.g., an attB site). In some cases, an attA site can be acceptor attachment site derived from a phage target sequence (e.g., an attP site).

In some cases, nucleic acid molecule including a guide sequence that is complementary to a target site and a nucleic acid sequence that encodes an attA site in a genome editing system provided herein can be engineered to include a nucleic acid sequence that encodes an attA site. For example, a nucleic acid sequence that encodes an attA site can be inserted into a nucleic acid using standard cloning or oligo capture techniques.

An attA site can be any appropriate length (e.g., can include any number of nucleotides). In some cases, an attA site can include from about 20 nucleotides to about 100 nucleotides (e.g., from about 20 nucleotides to about 90 nucleotides, from about 20 nucleotides to about 80 nucleotides, from about 20 nucleotides to about 70 nucleotides, from about 20 nucleotides to about 60 nucleotides, from about 20 nucleotides to about 50 nucleotides, from about 20 nucleotides to about 40 nucleotides, from about 20 nucleotides to about 30 nucleotides, from about 30 nucleotides to about 100 nucleotides, from about 40 nucleotides to about 100 nucleotides, from about 50 nucleotides to about 100 nucleotides, from about 60 nucleotides to about 100 nucleotides, from about 70 nucleotides to about 100 nucleotides, from about 80 nucleotides to about 100 nucleotides, from about 90 nucleotides to about 100 nucleotides, from about 30 nucleotides to about 90 nucleotides, from about 40 nucleotides to about 80 nucleotides, from about 50 nucleotides to about 70 nucleotides, from about 30 nucleotides to about 50 nucleotides, from about 40 nucleotides to about 60 nucleotides, from about 50 nucleotides to about 70 nucleotides, from about 60 nucleotides to about 80 nucleotides, or from about 70 nucleotides to about 90 nucleotides). For example, an attA site can include from about 25 nucleotides to about 45 nucleotides.

An attA site can include any appropriate nucleic acid sequence. Examples of attA sequences include, without limitation, nucleic acid sequences set forth in SEQ ID NOs: 11-84 and SEQ ID NO:254. In some cases, an attA site can have one or more amino acid modifications (e.g., one or more insertions, one or more deletions, and/or one or more substitutions) relative to an attA site described herein (e.g., SEQ ID NOs: 11-84 and SEQ ID NO:254), provided the attA site maintains the ability to be recognized and recombined by an integrase (e.g., a LSR). In some cases, an attA site can have at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, or 99%) sequence identity to a sequence set forth in any one of SEQ ID NOs: 11-84 and SEQ ID NO:254, provided that the attA site maintains the ability to be recognized and recombined by an integrase (e.g., a LSR).

In some cases, an attA sequence can be as described elsewhere (see, e.g., U.S. Serial No. 63/275,288, filed on November 3, 2021).

A system for stably integrating one or more nucleic acids into a target site within the genome of a cell provided herein can include any appropriate integrase. As used herein, the term “integrase” refers to a polypeptide that can recognize an attA site and an attD site and can meditate nucleic acid recombination between the attA site and the attD site. In some cases, an integrase can be a serine recombinase such as a large serine recombinase (LSR). In some cases, an integrase can be a landing pad integrase. In some cases, an integrase can be a genome-targeting integrase. In some cases, an integrase can be a multi-targeting integrase. In some cases, an integrase can be linked (e.g., covalently linked) to a polypeptide comprising a DNA binding domain and, optionally, a polymerase. For example, in some cases an integrase and a polypeptide comprising a DNA binding domain and, optionally, a polymerase can be provided together (e.g., as a fusion polypeptide comprising both the integrase and the polypeptide comprising a DNA binding domain and, optionally, a polymerase). In some cases when an integrase is linked to a polypeptide comprising a DNA binding domain and, optionally, a polymerase, the integrase can be linked directly to the polypeptide comprising a DNA binding domain and, optionally, a polymerase. In some cases when an integrase is linked to a polypeptide comprising a DNA binding domain and, optionally, a polymerase, the integrase can be linked to the polypeptide comprising a DNA binding domain and, optionally, a polymerase via a linker (e.g., a peptide linker).

In some cases, an integrase (e.g., serine recombinase such as a LSR) can include any appropriate amino acid sequence. For example, an integrase can have an amino acid sequence that includes one or more of the motifs set forth in SEQ ID NOs:233-245 (written in the common Prosite format). Examples of integrase sequences include, without limitation, amino acid sequences set forth in SEQ ID NOs:85-158. In some cases, an integrase can have one or more amino acid modifications (e.g., one or more insertions, one or more deletions, and/or one or more substitutions) relative to an integrase described herein (e.g., SEQ ID NOs: 85-158), provided the integrase maintains the ability to recognize and recombine an attA site and an attD site. In some cases, an integrase can have at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, or 99%) sequence identity to a sequence set forth in any one of SEQ ID NOs: 85- 158, provided that the integrase site maintains the ability to recognize and recombine an attA site and an attD site.

In some cases, an integrase (e.g., serine recombinase such as a LSR) can be as described elsewhere (see, e.g., U.S. Serial No. 63/275,288, filed on November 3, 2021).

A donor nucleic acid molecule including a nucleic acid cargo and an attD site in a system for stably integrating one or more nucleic acids into a target site within the genome of a cell provided herein can be any appropriate donor nucleic acid molecule. In some cases, a donor nucleic acid molecule can be a linear nucleic acid molecule. In some cases, a donor nucleic acid molecule can be a circular nucleic acid molecule (e.g., a plasmid or a minicircle).

A donor nucleic acid molecule can be any appropriate size (e.g., can include any number of nucleotides). In some cases, a donor nucleic acid molecule is from about 0.25 kb (250 nucleotides (nt)) to about 30 kb (e.g., from about 0.5 kb to about 30 kb, from about 1 kb to about 30 kb, from about 2 kb to about 30 kb, from about 5 kb to about 30 kb, from about 7 kb to about 30 kb, from about 10 kb to about 30 kb, from about 12 kb to about 30 kb, from about 15 kb to about 30 kb, from about 18 kb to about 30 kb, from about 20 kb to about 30 kb, from about 22 kb to about 30 kb, from about 25 kb to about 30 kb, from about 27 kb to about 30 kb, from about 0.25 kb to about 30 kb, from about 0.5 kb to about 25 kb, from about 1 kb to about 20 kb, from about 2 kb to about 15 kb, from about 5 kb to about 10 kb, from about 0.25 kb to about 25 kb, from about 0.25 kb to about 20 kb, from about 0.25 kb to about 15 kb, from about 0.25 kb to about 10 kb, from about 0.25 kb to about 7 kb, from about 0.25 kb to about 5 kb, from about 0.25 kb to about 3 kb, from about 0.25 kb to about 1 kb, from about 0.25 kb to about 0.5 kb, from about 0.25 kb to about 0.75 kb, from about 1 kb to about 5 kb, from about 2 kb to about 4 kb, from about 3 kb to about 7 kb, from about 5 kb to about 10 kb, from about 7 kb to about 12 kb, from about 12 kb to about 15 kb, from about 15 kb to about 18 kb, from about 18 kb to about 22 kb, from about 22 kb to about 25 kb, or from about 25 kb to about 28 kb). For example, a donor nucleic acid molecules can be from about 5kb to about 30 kb.

A donor nucleic acid molecule can include any appropriate nucleic acid cargo. A nucleic acid cargo can be any polynucleotide sequence that can be delivered to and inserted into a target site within the genome of a cell using a system for stably integrating one or more nucleic acids into a target site within the genome of a cell provided herein. In some cases, a nucleic acid cargo can include a nucleic acid encodes a gene product (e.g., a polypeptide or a non-coding RNA). For example, a nucleic acid cargo in a donor nucleic acid molecule of a system for stably integrating one or more nucleic acids into a target site within the genome of a cell provided herein can encode a polypeptide. Examples of polypeptides that can be encoded by a nucleic acid cargo in a donor nucleic acid molecule include, without limitation, detectable labels (e.g., peptide tags, fluorescent polypeptides, and enzymes), therapeutic polypeptides and biologically active fragments thereof (e.g., polypeptides useful for treating a diseases and/or condition) such as transcription factors, genome engineering systems, and polypeptides for eliciting an immune response, antibodies. For example, a nucleic acid cargo in a donor nucleic acid molecule of a system for stably integrating one or more nucleic acids into a target site within the genome of a cell provided herein can encode a RNA (e.g., a non- coding RNA). Examples of RNA that can be encoded by a nucleic acid cargo in a donor nucleic acid molecule include, without limitation, tRNA, rRNA, inhibitory RNAs (e.g., antisense RNAs, microRNAs (miRNAs), small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), and agomiRs), antagomiRs, aptamers, and long non-coding RNAs (IncRN As).

In cases where a donor nucleic acid molecule includes nucleic acid cargo that can encode a gene product, the donor nucleic acid also can include one or more regulatory elements operably linked to the nucleic acid encoding the gene product. Such regulatory elements can include promoter sequences, enhancer sequences, response elements, signal peptides, internal ribosome entry sequences, polyadenylation signals, terminators, and inducible elements that modulate expression (e.g., transcription or translation) of a nucleic acid. The choice of regulatory element(s) can depend on several factors, including, without limitation, inducibility, targeting, and the level of expression desired. For example, a promoter can be included in a donor nucleic acid molecule to facilitate transcription of a nucleic acid cargo encoding a gene product. A promoter can be a naturally occurring promoter or a recombinant promoter. A promoter can be ubiquitous or inducible (e.g., in the presence of tetracycline), and can affect the expression of a nucleic acid encoding a gene product in a general or tissue-specific manner. Examples of promoters include, without limitation, human ubiquitin C promoters, human synapsin 1 gene promoters, human glial fibrillary acidic protein promoters, promoters with tetracycline response elements, human elongation factor- 1 alpha promoters, cytomegalovirus promoters, CAG promoters, simian vacuolating virus 40 promoters, phosphoglycerate kinase gene promoters, and Ca ²⁺ /calmodulin-dependent protein kinase II promoters. As used herein, “operably linked” refers to positioning of a regulatory element in a donor nucleic acid molecule relative to a nucleic acid encoding a gene product in such a way as to permit or facilitate expression of the encoded gene product. For example, a donor nucleic acid molecule can contain a promoter and nucleic acid encoding a polypeptide. In this case, the promoter is operably linked to a nucleic acid encoding a polypeptide such that it drives expression of the polypeptide in cells. For example, a donor nucleic acid molecule can contain a promoter and nucleic acid encoding a non-coding RNA. In this case, the promoter is operably linked to a nucleic acid encoding a polypeptide such that it drives expression of the non-coding RNA in cells. In some cases, a donor nucleic acid molecule can include one or more additional nucleic acid elements. For example, a donor nucleic acid molecule can be flanked by inverted terminal repeats (ITRs; e.g., AAV ITRs).

In some cases, a donor nucleic acid molecule can include an attD site and, optionally, nucleic acid cargo that can encode a gene product, and can lack any other nucleic acid elements. For example, when a donor nucleic acid molecule is a plasmid, bacterial elements such as an origin of replication (Ori) site can be removed from the plasmid. For example, when a donor nucleic acid molecule is a plasmid, other coding sequences such as nucleic acid encoding a selectable marker such as an antibiotic resistance gene can be removed from the plasmid.

A donor nucleic acid molecule can include any appropriate attD site. In some cases, an attD site can be donor attachment site derived from a phage donor sequence (e.g., an attP site).

An attD site can be any appropriate length (e.g., can include any number of nucleotides). In some cases, an attD site can include from about 20 nucleotides to about 100 nucleotides (e.g., from about 20 nucleotides to about 90 nucleotides, from about 20 nucleotides to about 80 nucleotides, from about 20 nucleotides to about 70 nucleotides, from about 20 nucleotides to about 60 nucleotides, from about 20 nucleotides to about 50 nucleotides, from about 20 nucleotides to about 40 nucleotides, from about 20 nucleotides to about 30 nucleotides, from about 30 nucleotides to about 100 nucleotides, from about 40 nucleotides to about 100 nucleotides, from about 50 nucleotides to about 100 nucleotides, from about 60 nucleotides to about 100 nucleotides, from about 70 nucleotides to about 100 nucleotides, from about 80 nucleotides to about 100 nucleotides, from about 90 nucleotides to about 100 nucleotides, from about 30 nucleotides to about 90 nucleotides, from about 40 nucleotides to about 80 nucleotides, from about 50 nucleotides to about 70 nucleotides, from about 30 nucleotides to about 50 nucleotides, from about 40 nucleotides to about 60 nucleotides, from about 50 nucleotides to about 70 nucleotides, from about 60 nucleotides to about 80 nucleotides, or from about 70 nucleotides to about 90 nucleotides). For example, an attD site can include from about 25 nucleotides to about 45 nucleotides. An attD site can include any appropriate nucleic acid sequence. Examples of attD sequences include, without limitation, nucleic acid sequences set forth in SEQ ID NOs: 159- 232. In some cases, an attD site can have one or more amino acid modifications (e.g., one or more insertions, one or more deletions, and/or one or more substitutions) relative to an attD site described herein (e.g., SEQ ID NOs: 159-232), provided the attD site maintains the ability to be recognized and recombined by an integrase (e.g., an LSR). In some cases, an attD site can have at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, or 99%) sequence identity to a sequence set forth in any one of SEQ ID NOs: 159-232, provided that the attD site maintains the ability to be recognized and recombined by an integrase (e.g., a LSR).

In some cases, an attD sequence can be as described elsewhere (see, e.g., U.S. Serial No. 63/275,288, filed on November 3, 2021).

Also provided herein are methods for using systems for stably integrating one or more nucleic acids into a target site within the genome of a cell provided herein (e.g., systems including (a) a genome-editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site). In some cases, a system for stably integrating one or more nucleic acids into a target site within the genome of a cell provided herein can be delivered to a cell to stably integrate a nucleic acid into the genome of the cell. For example, a system including (a) a genome-editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site can be delivered to a cell to stably integrate the nucleic acid cargo into the genome of the cell. In some cases, the components of a system for stably integrating one or more nucleic acids into a target site within the genome of a cell provided herein can be delivered to a cell in vitro. In some cases, the components of a system for stably integrating one or more nucleic acids into a target site within the genome of a cell provided herein can be delivered to a cell ex vivo. In some cases, the components of a system for stably integrating one or more nucleic acids into a target site within the genome of a cell provided herein can be delivered to a cell in vivo. Any appropriate method can be used to deliver components of a system for stably integrating one or more nucleic acids into a target site within the genome of a cell provided herein (e.g., systems including (a) a genome-editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site) to cells (e.g., cells within a living mammal). In some cases, a genome-editing system that can insert an attA into a target site within a genome can be delivered to a cell as a complex including (i) a polypeptide having a DNA binding domain and, optionally, a polymerase and (ii) a nucleic acid molecule including a guide sequence that is complementary to the target site and a nucleic acid sequence that encodes an attA site. In some cases, a genome-editing system that can insert an attA into a target site within a genome can be delivered to a cell as a nucleic acid encoding the genome-editing system (e.g., a vector designed to express the genome-editing system) such that a complex including (i) a polypeptide having a DNA binding domain and, optionally, a polymerase and (ii) a nucleic acid molecule including a guide sequence that is complementary to the target site and a nucleic acid sequence that encodes an attA site is formed within the cell. In some cases, an integrase that can target the attA site and the attD site can be delivered to a cell as a polypeptide. In some cases, an integrase that can target the attA site and the attD site can be delivered to a cell as a nucleic acid encoding the integrase (e.g., a vector designed to express the integrase). In some cases, a donor nucleic acid molecule including a nucleic acid cargo and an attD site can be delivered to a cell as a linear nucleic acid molecule. In some cases, a donor nucleic acid molecule including a nucleic acid cargo and an attD site can be delivered to a cell as a circular nucleic acid (e.g., a vector). For example, a genome-editing system that can insert an attA into a target site within a genome and an integrase that can target the attA site and the attD site can be delivered to a cell as polypeptides, and a donor nucleic acid molecule including a nucleic acid cargo and an attD site are administered to cell can be delivered to the cell in the form of a vector (e.g., a non-viral vector). In some cases, nucleic acid encoding a genome-editing system that can insert an attA into a target site within a genome, nucleic acid encoding an integrase that can target the attA site and the attD site, and a donor nucleic acid molecule including a nucleic acid cargo and an attD site can be delivered to a cell in the form of one or more vectors (e.g., one or more viral vectors and/or one or more non- viral vectors).

When a vector used to deliver nucleic acid encoding a genome-editing system that can insert an attA into a target site within a genome, nucleic acid encoding an integrase that can target the attA site and the attD site, and/or a donor nucleic acid molecule including a nucleic acid cargo and an attD site is a viral vector, any appropriate viral vector can be used. A viral vector can be derived from a positive-strand virus or a negative-strand virus. A viral vector can be derived from a virus with a DNA genome or a RNA genome. In some cases, a viral vector can be a chimeric viral vector. In some cases, a viral vector can infect dividing cells. In some cases, a viral vector can infect non-dividing cells. Examples of virus-based vectors that can be used to deliver nucleic acid encoding a genome-editing system that can insert an attA into a target site within a genome, nucleic acid encoding an integrase that can target the attA site and the attD site, and/or a donor nucleic acid molecule including a nucleic acid cargo and an attD site include, without limitation, virus-based vectors based on adenoviruses, adeno-associated viruses (AAVs), Sendai viruses, retroviruses, or lentiviruses. In some cases, a donor nucleic acid molecule including a nucleic acid cargo and an attD site can be delivered on an AAV.

When a vector used to deliver nucleic acid encoding a genome-editing system that can insert an attA into a target site within a genome, nucleic acid encoding an integrase that can target the attA site and the attD site, and/or a donor nucleic acid molecule including a nucleic acid cargo and an attD site is a non-viral vector, any appropriate non-viral vector can be used. In some cases, a non-viral vector can be an expression plasmid (e.g., a cDNA expression vector).

When nucleic acid encoding a genome-editing system that can insert an attA into a target site within a genome and/or nucleic acid encoding an integrase is delivered to a cell, the nucleic acid can be used for transient expression of a genome-editing system and/or an integrase or for stable expression of a genome-editing system and/or an integrase.

In cases where a nucleic acid encoding a genome-editing system that can insert an attA into a target site within a genome and/or nucleic acid encoding an integrase is used to deliver a genome-editing system and/or an integrase to a cell, the nucleic acid also can include one or more regulatory elements operably linked to the nucleic acid encoding the genome-editing system and/or the integrase. Such regulatory elements can include promoter sequences, enhancer sequences, response elements, signal peptides, internal ribosome entry sequences, polyadenylation signals, terminators, and inducible elements that modulate expression (e.g., transcription or translation) of a nucleic acid. The choice of regulatory element(s) can depend on several factors, including, without limitation, inducibility, targeting, and the level of expression desired. For example, a promoter can be included in a nucleic acid encoding a genome-editing system that can insert an attA into a target site within a genome and/or nucleic acid encoding an integrase to facilitate transcription of the genome- editing system and/or the integrase. A promoter can be a naturally occurring promoter or a recombinant promoter. A promoter can be ubiquitous or inducible (e.g., in the presence of tetracycline), and can affect the expression of a nucleic acid encoding a gene product in a general or tissue-specific manner. Examples of promoters include, without limitation, human ubiquitin C promoters, human synapsin 1 gene promoters, human glial fibrillary acidic protein promoters, promoters with tetracycline response elements, human elongation factor- 1 alpha promoters, cytomegalovirus promoters, CAG promoters, simian vacuolating virus 40 promoters, phosphoglycerate kinase gene promoters, and Ca ²⁺ /calmodulin-dependent protein kinase II promoters. As used herein, “operably linked” refers to positioning of a regulatory element in a donor nucleic acid molecule relative to a nucleic acid encoding a genome- editing system that can insert an attA into a target site within a genome and/or nucleic acid encoding an integrase in such a way as to permit or facilitate expression of the encoded genome-editing system and/or the encoded integrase. For example, a nucleic acid encoding a genome-editing system that can insert an attA into a target site within a genome can contain a promoter and nucleic acid encoding a genome- editing system. In this case, the promoter is operably linked to a nucleic acid encoding a genome-editing system that can insert an attA into a target site within a genome such that it drives expression of the genome- editing system in cells. For example, a nucleic acid encoding an integrase can contain a promoter and nucleic acid encoding the integrase. In this case, the promoter is operably linked to a nucleic acid encoding an integrase such that it drives expression of the integrase in cells. In some cases, the components of a system for stably integrating one or more nucleic acids into a target site within the genome of a cell provided herein (e.g., systems including (a) a genome- editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site) can be delivered to cells (e.g., cells within a living mammal) at the same time. For example, a system for stably integrating one or more nucleic acids into a target site within the genome of a cell can be delivered to a cell in a single composition containing (a) a genome-editing system that can insert an attA into a target site within a genome (or nucleic acid encoding such a genome-editing system), (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site (or nucleic acid encoding such an integrase). For example, a system for stably integrating one or more nucleic acids into a target site within the genome of a cell can be delivered to a cell in a single composition containing (a) a genome-editing system that can insert an attA into a target site within a genome linked (e.g., covalently linked as a fusion polypeptide) to (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and containing (c) an integrase (e.g., a LSR) that can target the attA site and the attD site. For example, a system for stably integrating one or more nucleic acids into a target site within the genome of a cell can be delivered to a cell in a single composition containing a nucleic acid encoding a polypeptide (e.g., a fusion polypeptide) including both a genome-editing system that can insert an attA into a target site within a genome linked and an integrase (e.g., a LSR) that can target the attA site and an attD site, and a donor nucleic acid molecule including a nucleic acid cargo and the attD site.

In some cases, the components of a system for stably integrating one or more nucleic acids into a target site within the genome of a cell provided herein (e.g., systems including (a) a genome- editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site) can be delivered to cells (e.g., cells within a living mammal) independently. For example, a system for stably integrating one or more nucleic acids into a target site within the genome of a cell can be delivered to a cell as in a first composition containing (a) a genome-editing system that can insert an attA into a target site within a genome (or nucleic acid encoding such a genome-editing system), and a second composition containing (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site (or nucleic acid encoding such an integrase). For example, a system for stably integrating one or more nucleic acids into a target site within the genome of a cell can be delivered to a cell as in a first composition containing (a) a genome-editing system that can insert an attA into a target site within a genome (or nucleic acid encoding such a genome- editing system) and (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and a second composition containing (c)an integrase (e.g., a LSR) that can target the attA site and the attD site (or nucleic acid encoding such an integrase). For example, a system for stably integrating one or more nucleic acids into a target site within the genome of a cell can be delivered to a cell as in a first composition containing (a) a genome-editing system that can insert an attA into a target site within a genome (or nucleic acid encoding such a genome-editing system), a second composition containing (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and a third composition containing (c) an integrase (e.g., a LSR) that can target the attA site and the attD site (or nucleic acid encoding such an integrase).

In some cases, the methods and materials provided herein (e.g., systems including (a) a genome- editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site) can be used for labelling a gene product (e.g., a polypeptide or a non-coding RNA) within a cell (e.g., a plant cell or a mammalian cell). For example, the methods and materials provided herein can be used to label a gene product encoded by an endogenous nucleic acid within a cell (e.g., a prokaryotic cell or a eukaryotic cell such as a plant cell or an animal cell). In some cases, a gene product within a cell can be labeled by delivering a system for stably integrating one or more nucleic acids into a target site within the genome of a cell provided herein (e.g., a system including (a) a genome- editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site) to a cell (e.g., a plant cell or a mammalian cell) to stably integrate a nucleic acid encoding a detectable label in-frame with an endogenous nucleic acid encoding a target gene product such that the encoded target gene product is fused to the detectable label. For example, (a) a genome-editing system that can insert an attA into a target site within a genome that is in-frame with an endogenous nucleic acid encoding a target gene product, (b) a donor nucleic acid molecule including a nucleic acid cargo encoding a detectable label and an attD site, and (c) an integrase that can target the attA site and the attD site can be delivered to a cell to stably integrate the nucleic acid cargo encoding the detectable label into the genome such that the encoded target gene product is fused to the detectable label.

When a nucleic acid cargo encoding a detectable label is stably integrated into the genome of a cell (e.g., a plant cell or a mammalian cell) to label a target polypeptide within the cell, any appropriate detectable label can be used. Examples of detectable labels include, without limitation, luminescent tags (e.g., HiBiT), peptide tags (e.g., HaloTag, Flag tags, HA tags, MS2/PP7 tags, Sun/Moon tags, and poly(His) tags), fluorescent polypeptides (e.g., mCherry and green fluorescent polypeptides (GFPs; e.g., mNeonGreen)), and enzymes (e.g., glutathione-S-transferases (GSTs), luciferases, horseradish peroxidases (HRPs), alkaline phosphatases (APs), and apurinic/apyrimidinic endodeoxyribonuclease 2 (APEX2) polypeptides).

In some cases, a nucleic acid cargo encoding a detectable label can be integrated into the genome upstream of an endogenous nucleic acid encoding a target polypeptide such that the detectable label is fused to the N-terminus of the target polypeptide.

In some cases, a nucleic acid cargo encoding a detectable label can be integrated into the genome downstream of an endogenous nucleic acid encoding a target polypeptide such that the detectable label is fused to the C-terminus of the target polypeptide.

In some cases, the methods and materials provided herein (e.g., systems including (a) a genome- editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site) can be used to increase expression of a polypeptide within a cell (e.g., a plant cell or a mammalian cell). For example, the methods and materials provided herein can be used to increase expression of a polypeptide encoded by an endogenous nucleic acid within a cell (e.g., a prokaryotic cell or a eukaryotic cell such as a plant cell or an animal cell). In some cases, expression of a polypeptide within a cell can be increased by delivering a system for stably integrating one or more nucleic acids into a target site within the genome of a cell provided herein (e.g., a system including (a) a genome-editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site) to a cell (a plant cell or a mammalian cell) to stably integrate a regulatory element (e.g., a promoter sequence) near (e.g., upstream of) an endogenous nucleic acid encoding a target polypeptide such that the regulatory element is operably linked to and increases expression of the encoded target polypeptide. For example, (a) a genome-editing system that can insert an attA into a target site within a genome near an endogenous nucleic acid encoding a target polypeptide, (b) a donor nucleic acid molecule including a nucleic acid cargo containing a promoter sequence and an attD site, and (c) an integrase that can target the attA site and the attD site can be delivered to a cell to stably integrate the promoter sequence into the genome such that the expression of the encoded target polypeptide is increased.

In some cases, the methods and materials provided herein (e.g., systems including (a) a genome- editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site) can be used for making a transgenic organism (e.g., a non-human transgenic organism). For example, the methods and materials provided herein can be used to express an exogenous polypeptide within a cell such as a eukaryotic cell. In some cases, the methods and materials provided herein can be used to stably integrate a transgene (e.g., a transgene encoding an exogenous polypeptide) into the genome of a cell (e.g., an embryonic stem cell) that can give rise to an animal (e.g., a non- human animal). In some cases, the methods and materials provided herein can be used to stably integrate a transgene (e.g., a transgene encoding an exogenous polypeptide) into the genome of a cell (e.g., a plant cell) that can give rise to a plant. In some cases, a transgenic organism (e.g., a non- human transgenic organism) can be created by delivering a system for stably integrating one or more nucleic acids into a target site within the genome of a cell provided herein (e.g., a system including (a) a genome- editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site) to a cell (e.g., a plant cell or a non-human embryonic stem cell) to stably integrate a transgene (e.g., a transgene encoding a polypeptide of interest) into the genome such that the transgene is expressed by the cell. For example, (a) a genome- editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a transgene and an attD site, and (c) an integrase that can target the attA site and the attD site can be delivered to a cell to stably integrate the transgene into the genome such that the transgene is expressed by the cell.

In some cases, the methods and materials provided herein (e.g., systems including (a) a genome- editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site) can be used for making a transgenic cell (e.g., a transgenic immune cell such as a transgenic T cell, a transgenic NK cell, or a transgenic macrophage) having (e.g., engineered to have) a receptor (e.g., a T cell receptor (TCR); a NK cell receptor (NKR), or a chimeric antigen receptor (CAR)). For example, (a) a genome-editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a transgene encoding a CAR and an attD site, and (c) an integrase that can target the attA site and the attD site can be delivered to a T cell (e.g., an ex vivo human T cell) to stably integrate the transgene into the genome of the T cell such that the CAR is expressed by the T cell (e.g., to generate a CAR T cell). For example, (a) a genome-editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a transgene encoding a TCR (e.g., a wild type TCR or an engineered TCR) and an attD site, and (c) an integrase that can target the attA site and the attD site can be delivered to an NK cell (e.g., an ex vivo human NK cell) to stably integrate the transgene into the genome of the NK cell such that the TCR is expressed by the NK cell (e.g., to generate an NK cell expressing the TCR). For example, (a) a genome- editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a transgene encoding a NKR (e.g., a wild type NKR or an engineered NKR) and an attD site, and (c) an integrase that can target the attA site and the attD site can be delivered to an NK cell (e.g., an ex vivo human NK cell) to stably integrate the transgene into the genome of the NK cell such that the NKR is expressed by the NK cell (e.g., to generate an NK cell expressing the NKR). Any appropriate receptor (e.g., any appropriate TCR, any appropriate NKR, or any appropriate CAR) can be integrated into the genome of a cell (e.g., an immune cell such as a T cell or a NK cell) as described herein. In some cases, a CAR can be as described elsewhere (e.g., De Bousser et al., Cancers (Basel), 13(23):6067 (2021); Eyquem et al., Nature, 543(7643):113-117 (2017); and Larson et al., Nat. Rev. Cancer, 21(3): 145-161 (2021)).

In some cases, the methods and materials provided herein (e.g., systems including (a) a genome- editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site) can be used for making a transgenic plant having (e.g., engineered to have) pathogen resistance (e.g., bacterial resistance or viral resistance). For example, (a) a genome-editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a transgene encoding a pathogen resistance polypeptide and an attD site, and (c) an integrase that can target the attA site and the attD site can be delivered to a plant cell to stably integrate the transgene into the genome such that the pathogen resistance polypeptide is expressed by the cell. Any appropriate pathogen resistance polypeptide can be integrated into a plant cell genome to create a pathogen resistant transgenic plant as described herein. In some cases, a pathogen resistance polypeptide can be as described elsewhere (e.g., Dong et al., Plant Physiol., 180(l):26-38 (2019)).

In some cases, the methods and materials provided herein (e.g., systems including (a) a genome- editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site) can be used for making a transgenic plant having (e.g., engineered to have) herbicide resistance. For example, (a) a genome-editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a transgene encoding a herbicide resistance polypeptide and an attD site, and (c) an integrase that can target the attA site and the attD site can be delivered to a plant cell to stably integrate the transgene into the genome such that the herbicide resistance polypeptide is expressed by the cell. Any appropriate herbicide resistance polypeptide can be integrated into a plant cell genome to create an herbicide resistant transgenic plant as described herein. In some cases, an herbicide resistance polypeptide can be as described elsewhere (e.g., Sun et al., Molecular Plant, 9.4:628-631 (2016); Li et al., Nature Plants, 2: 16139 (2016); Tatsis et al., Curr. Opin. Biotech., 42: 126-132 (2016); Ducat et al., Curr. Opin. Chem. Biol., 16(3-4):337-344 (2012); Sanghera et al., Curr. Genomics., 12(l):30-43 (2011); Dong et al., Nat. Commun., 11: 1178 (2020); and Lu et al., Nat.

Biotechnol., 38: 1402-1407 (2020)).

In some cases, the methods and materials provided herein (e.g., systems including (a) a genome- editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site) can be used for making an organism (e.g., a non- human organism) having reduced or eliminated levels of a polypeptide (e.g., a non-human knock-out organism). For example, the methods and materials provided herein can be used to disrupt and/or replace an endogenous nucleic acid encoding a target polypeptide within a cell such as a eukaryotic cell. In some cases, the methods and materials provided herein can be used to stably integrate a nucleic acid molecule (e.g., knock-out cassette) into the genome of a cell (e.g., an embryonic stem cell) that can give rise to an organism (e.g., a non-human animal) to disrupt and/or replace an endogenous nucleic acid encoding a target polypeptide. In some cases, the methods and materials provided herein can be used to stably integrate a nucleic acid molecule (e.g., knock-out cassette) into the genome of a cell (e.g., a plant cell) that can give rise to a plant to disrupt and/or replace an endogenous nucleic acid encoding a target polypeptide.

In some cases, an endogenous nucleic acid encoding a target polypeptide within a cell can be disrupted and/or replaced by delivering a system for stably integrating one or more nucleic acids into a target site within the genome of a cell provided herein (e.g., a system including (a) a genome-editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site) to a cell (a plant cell or a mammalian cell) to stably integrate a nucleic acid molecule within an endogenous nucleic acid encoding a target polypeptide such that the nucleic acid molecule disrupts and/or replaces the endogenous nucleic acid encoding a target polypeptide and expression of the endogenous nucleic acid encoding the target polypeptide is reduced or eliminated. For example, (a) a genome-editing system that can insert an attA into a target site within a genome that is in-frame with an endogenous nucleic acid encoding a target polypeptide, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase that can target the attA site and the attD site can be delivered to a cell to stably integrate the nucleic acid cargo into the genome such that the nucleic acid cargo disrupts and/or replaces an endogenous nucleic acid encoding a target polypeptide such that the nucleic acid molecule disrupts and/or replaces the endogenous nucleic acid encoding a target polypeptide and expression of the encoded target polypeptide is reduced or eliminated.

In some cases, a nucleic acid cargo that can be stably integrated into a genome of a cell (e.g., a non-human animal cell or a plant cell) to disrupt and/or replace an endogenous nucleic acid encoding a target polypeptide such that expression of the encoded the target polypeptide is reduced or eliminated can include a stop codon.

In some cases, a nucleic acid cargo that can be stably integrated into a genome of a cell (e.g., a non-human animal cell or a plant cell) to disrupt and/or replace an endogenous nucleic acid encoding a target polypeptide such that expression of the encoded the target polypeptide is reduced or eliminated can include a splice acceptor site.

In some cases, a nucleic acid cargo that can be stably integrated into a genome of a cell (e.g., a non-human animal cell or a plant cell) to disrupt and/or replace an endogenous nucleic acid encoding a target polypeptide such that expression of the encoded the target polypeptide is reduced or eliminated can include nucleic acid encoding a selectable marker such that the selectable marker is expressed by the cell. For example, a nucleic acid cargo can be stably integrated into a genome of a cell such that the selectable marker is under the control of the regulatory elements for the disrupted and/or replaced endogenous nucleic acid encoding a target polypeptide.

In some cases, a nucleic acid cargo that can be stably integrated into a genome of a cell (e.g., a non-human animal cell or a plant cell) to disrupt and/or replace an endogenous nucleic acid encoding a target polypeptide such that expression of the encoded the target polypeptide is reduced or eliminated can include a detectable label such that the detectable label is expressed by the cell. For example, a nucleic acid cargo can be stably integrated into a genome of a cell such that the detectable label is under the control of the regulatory elements for the disrupted and/or replaced endogenous nucleic acid encoding a target polypeptide.

In some cases, the methods and materials provided herein (e.g., systems including (a) a genome- editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site) can be used for treating a mammal (e.g., a human) having a disease or disorder. For example, (a) a genome-editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a transgene encoding a therapeutic gene product and an attD site, and (c) an integrase that can target the attA site and the attD site can be delivered to a cell to stably integrate the transgene into the genome such that the therapeutic gene product is expressed by the cell. In some cases, the methods and materials provided herein can be used to treat a mammal (e.g., a human) have a disease or disorder associated with reduced or eliminated levels of a gene product (e.g., reduced or eliminated levels of a polypeptide or reduced or eliminated levels of a non-coding RNA). In some cases, the methods and materials provided herein can be used to treat a mammal (e.g., a human) have a disease or disorder associated with a mutated gene product (e.g., a mutated polypeptide or a mutated non-coding RNA).

When the methods and materials provided herein are used to treat a mammal, the mammal can be any appropriate mammal. Examples of mammals that can be treated as described herein include, without limitation, humans, non-human primates such as chimpanzees and monkeys, dogs, cats, horses, cows, pigs, sheep, mice, rats, rabbits, guinea pigs, birds, fish, (e.g., zebrafish (Danio rerio), medaka (Oryzias Latipes), and turquoise killifish (Nothobranchius fii zeri)). nematodes (e.g., Caenorhabditis elegans), and flies (e.g., Drosophila melanogaster).

In some cases when treating a mammal as described herein, the components of a system for stably integrating one or more nucleic acids into a target site within the genome of a cell provided herein (e.g., systems including (a) a genome-editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site) can be delivered to cells within a living mammal (e.g., can be delivered to in vivo cells).

In some cases when treating a mammal as described herein, the components of a system for stably integrating one or more nucleic acids into a target site within the genome of a cell provided herein (e.g., systems including (a) a genome-editing system that can insert an attA into a target site within a genome, (b) a donor nucleic acid molecule including a nucleic acid cargo and an attD site, and (c) an integrase (e.g., a LSR) that can target the attA site and the attD site) can be delivered to cells obtained from a mammal (e.g., can be delivered to ex vivo cells), and then the cells containing the stably integrated nucleic acid can be administered to the mammal to be treated. In some cases, the components of a system for stably integrating one or more nucleic acids into a target site within the genome of a cell provided herein are delivered ex vivo to cell obtained from the mammal to be treated (e.g., an autologous cell). In some cases, the components of a system for stably integrating one or more nucleic acids into a target site within the genome of a cell provided herein are delivered ex vivo to cell obtained from a donor mammal (e.g., an allogeneic cell).

Any appropriate transgene encoding a therapeutic gene product can be integrated into a cell genome to treat a mammal as described herein. Examples of therapeutic gene products include, without limitation, adenosine deaminase (e.g., to treat a mammal having severe combined immunodeficiency (SCID)), α-1 antitrypsin (e.g., to treat a mammal having liver damage such as cirrhosis), cystic fibrosis transmembrane conductance regulator (CFTR; e.g., to treat a mammal having cystic fibrosis (CF)), β-hemoglobin (HBB; e.g., to treat a mammal having thalassemia), oculocutaneous albinism II (0CA2; e.g., to treat a mammal having oculocutaneous albinism (OCA), Huntingtin (HTT; e.g., to treat a mammal having Huntington's disease), dystrophia myotonica-protein kinase (DMPK; e.g., to treat a mammal having myotonic dystrophy 1 (DM1)), low-density lipoprotein receptor (LDLR; e.g., to treat a mammal having familial hypercholesterolemia (FH)), apolipoprotein B (APOB; e.g., to treat a mammal having FH), neurofibromin 1 (NF1; e.g., to treat a mammal having neurofibromatosis), polycystic kidney disease 1 (PKD1; e.g., to treat a mammal having polycystic kidney disease), polycystic kidney disease 2 (PKD2; e.g., to treat a mammal having polycystic kidney disease), coagulation factor VIII (F8; e.g., to treat a mammal having hemophilia), dystrophin (DMD; e.g., to treat a mammal having Duchenne muscular dystrophy (DMD)), phosphate-regulating endopeptidase homologue X-linked (PHEX; e.g., to treat a mammal having hypophosphatemic rickets), methyl-CpG-binding protein 2 (MECP2; e.g., to treat a mammal having Rett Syndrome), ubiquitin-specific peptidase 9Y, Y-linked (USP9Y; e.g., to treat a mammal having spermatogenic failure), a carbamoyl-phosphate synthase 1 (CPS1) polypeptide, an ATP binding cassette subfamily A member 4 (ABCA4) polypeptide, an fatty acid elongase 4 (ELOVL) polypeptide, amyosin VIIA (MY07A) polypeptide, an usher syndrome 1C (USH1C) polypeptide, a cadherin related 23 (CDH23) polypeptide, a protocadherin related 15 (PCDH15) polypeptide, an usher syndrome 1G (USH1G) polypeptide, an usher syndrome 2A (USH2A) polypeptide, an adhesion G protein- coupled receptor VI (ADGRV1) polypeptide, a whirlin (WHRN) polypeptide, a clarin 1 (CLRN1) polypeptide, a retinitis pigmentosa 1 (RP1) polypeptide, an eyes shut homolog (EYS) polypeptide, a lipoprotein (a) (LPA) polypeptide, a lipoprotein lipase (LPL) polypeptide, an apolipoprotein C2 (AP0C2) polypeptide, an apolipoprotein A5 (AP0A5) polypeptide, a lipase maturation factor 1 (LMF1) polypeptide, a glycosylphosphatidylinositol anchored high density lipoprotein binding protein 1 (GPIHBP1) polypeptide, a proprotein convertase subtilisin/kexin type 9 (PCSK9) polypeptide, a ryanodine receptor 2 (RYR2) polypeptide, a calsequestrin 2 (CASQ2) polypeptide, a myosin heavy chain 7 (MYH7) polypeptide, a myosin binding protein C3 (MYBPC3) polypeptide, a troponin T2, cardiac type (TNNT2) polypeptide, and a troponin 13, cardiac type (TNNI3) polypeptide, and C9orf72 polypeptide (e.g., to treat a mammal having C9orf72 amyotrophic lateral sclerosis and frontotemporal dementia (C9 ALS/FTD)). In some cases, a therapeutic gene product can be as described elsewhere (e.g., Suzuki et al., Mol. Then, 28.7:1684-1695 (2020); Pierce et al., Cold Spring Harbor Perspect. Med. 5:9 a017285 (2015); Urnov et al., Nature, 435.7042:646-651 (2005); Phelps et al., Human Mol. Gen., 4.8:1251-1258 (1995); and Ellerby et al., Neurotherapeutics, 16(4): 924-927 (2019)).

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES

Example 1: Stable Integration of Multi-Kilobase DNA Cargos Into Eukaryotic Cell Genomes

Large serine recombinases (LSRs) are a family of enzymes encoded in phage genomes that site-specifically and unidirectionally recombine short DNA attachment sites present on phage and bacterial genome, resulting in integration of the multi-kilobase phage genome into the bacterial genome.

This Example describes the utilization of a prime editor in combination with a LSR for programmable recombination of multiple kilobase cargo into the genome. For example, a prime editor can be used to insert an attA site into a desired genomic context, and a LSR can integrate a nucleic acid cargo into the target site. Schematic images of exemplary methods of using a prime editor in combination with a LSR for programmable recombination of multiple kilobase cargo into the genome are shown in Figure 1.

Methods

Cloning of pegRNAs and ngRNAs

For pegRNAs, spacer sequences, extension templates, and SpCas9 sgRNA scaffold sequences were synthesized (Integrated DNA Technologies) and cloned via ligation of annealed oligonucleotides into BsmBI digested acceptor vector (pU6-pegRNA-GG-acceptor, Addgene plasmid no. 132777). For ngRNAs, spacers were synthesized (Integrated DNA Technologies) and cloned via ligation of annealed oligonucleotides into BbsI digested acceptor vector (pCB007 SpCas9_sgRNA_cloning_Backbone). Cell lines and cell culture

Experiments were carried out in HEK-293FT cells (Thermo Fisher). HEK-293FT cells were grown in DMEM (Gibco) media supplemented with 10% FBS (Hyclone), penicillin (10,000 I.U./mL), and streptomycin (10,000 ug/mL).

Prime Editing Transfection

20,000 HEK293FT cells were plated into poly-D-lysine coated 96 well plates. One day later, 250ng prime editor plasmid (pCMV-PE2-P2A-GFP Addgene plasmid #132776), 83 ng pegRNA plasmid, and 27.6 ng ngRNA plasmid were transfected into the cells using Lipofectamine 2000 (Thermo). 3 days later, cells were extracted with DNA QuickExtract (Lucigen). Edits were verified via PCR (Platinum Superfi PCR Master Mix, Thermo) across the edited locus. Sanger sequencing was analyzed with ICE analysis (Synthego) to determine the percentage of cells containing the edit.

2-step transfection

Trans delivery. Prime editor, LSR and guide RNAs were transfected into HEK293FT cells in a single step or two step transfection. For two-step transfections, 20,000 HEK293FT cells were plated into poly-D-lysine coated 96 well plates. One day later, 250ng prime editor plasmid , 83ng pegRNA, and 27.6ng ngRNA were transfected into the cells using Lipofectamine 2000 (Thermo). Two days later, 200ng LSR effector plasmid and lOOng attD donor plasmid were transfected into the cells using Lipofectamine 2000 (Thermo). Cells were harvested two days later using DNA QuickExtract (Lucigen). Prime editing and LSR mediated donor integration were confirmed using PCR (Platinum Superfi PCR Master Mix, Thermo Fisher) across the insertion junction. For one-step transfections, the same quantities of Prime editor, ngRNA, pegRNA, LSR, and donor plasmid were co-transfected on day 0, and cells were harvested on day 5 for PCR.

Sanger sequencing validation of donor integration. The Prime editing elements are transfected, and two days later the LSR and donor DNA are delivered. 4 days post- transfection, the gDNA is extracted, purified, and PCR and Sanger sequencing is performed across the donor-genome junction. Cloning PE-LSR Effector Plasmid

Prime editing plasmid (pCMV-PE2, Addgene Plasmid #132775) was modified with gibson cloning to include an XTEN 48 linker, a L139P mutation in the MMuLV RT, and either a (GGS)6 (for cis LSR delivery) or a self-cleavable P2A (for trans LSR delivery) linker and BsmbI golden gate landing pad at the C terminus of the RT. Human codon optimized LSRs were cloned into the BsmBI landing pad via golden gate assembly.

1-step transfection and integration detection

Three plasmids containing the effector, donor, and guides are co-transfected into mammalian cells (HEK293FT). Three days later, gDNA is extracted, purified, and donor integration is determined by qPCR and ddPCR of the donor-genome junction.

1-step prime editing, 1 pegRNA

20,000 HEK293FT cells were plated into poly-D-lysine coated 96 well plates. One day later, 375ng effector plasmid, lOOng pegRNA, and 50ng ngRNA were transfected into the cells using Lipofectamine 2000 (Thermo). After 72 hours, media was removed and cells were resuspended in 40uL DNA QuickExtract (Lucigen). Next, the cells were transferred to a PCR plate, and incubated at 65°C for 15 minutes, 68°C for 15 minutes, and 98°C for 10 minutes. Finally, samples were purified with 0.9X Ampure XP beads (Beckman Coulter).

1-step prime editing, 2 pegRNAs

Cells were plated as previously described and transfected with lipofectamine 2000, delivering 375 ng effector plasmid, 60 ng of each twinPE pegRNA, and 250 ng cargo plasmid. 72 hrs post transfection, cells were harvested and purified with DNA Quick Extract and Ampure XP beads. qPCR verification of targeted recombination. qPCR primers and a FAM probe (IDT and Elim Bio) were designed to amplify the integration junction. As a genomic DNA reference, qPCR primers and a HEX probe (IDT and Elim Bio) were designed to amplify a non-edited region of the ACTB gene. IOUL qPCR reactions were performed with 5uL Taqman Fast Advanced 2x Master Mix, 250nM of each primer, 200nM of each probe, and luL of extracted genomic DNA. qPCR was run on the 480 LightCycler (Roche), which calculated Ct values. Delta Ct indicates the difference between the Ct of the integration and reference probe Ct values. ddPCR of donor integration

To quantify integration efficiency by digital droplet PCR, 20uL solutions were prepared containing 1 OuL 2x ddPCR Supermix for Probes (Bio-Rad), 900nM primers, 250nM probes, 0.2uL Sad restriction enzyme, and 1 uL genomic DNA. Identical primers and probes were used as the set used for qPCR. the 20uL reaction was transferred to a Dg8 Cartridge (Bio-Rad) with 70uL Droplet Generation oil for Probes (Bio-Rad), and loaded into a QX2000 droplet generator (Bio-Rad). 40uL of the droplets were transferred to a 96 well plate and thermocycled according to manufacturer’s specifications. Finally, the plate was loaded into the QX200 droplet reader (Bio-Rad) for droplet analysis and copy number quantification.

Prime edit detection

To determine efficiency of prime editing alone, identical transfection conditions are carried out, but without the donor plasmid with a stuffer plasmid in its place (pucl9). Three days post transfection, gDNA was extracted and purified as described above, and the edited locus is sequenced via next generation sequencing on an Illumina Miseq.

Results

Validation of Prime Editing attA

Three days after transfecting cells with plasmids encoding the prime editor, pegRNA, and ngRNA, gDNA was extracted and PCR was performed on target locus (HEK3). Sanger sequencing and ICE analysis confirmed that the attA for Bxbl and PaOl, which is encoded on the pegRNA, can be integrated into the target locus (Figure 3).

PCR validation of donor integration

To directly detect installation of the attachment site at the target locus and integration of cargo into the attachment site, PCRs were performed across the integration junction. Via gel electrophoresis (Figure 4) and Sanger sequencing of PCR products (Figures 5A and 5B), on- target donor integration mediated by the Bxbl and PaOl LSR-PE system was confirmed.

Evaluation of attA length

Truncation of attA site increased prime editing efficiency, but decreased LSR integration efficiency (Figure 6). qPCR of donor integration, 1 step delivery, 1 pegRNA

Via qPCR, we confirmed integration of the donor plasmid into the target loci for both LMNB1 and ACTB targeting pegRNAs, and utilizing Nm60, Kp03, Si74, and PaOl as the recombinase in the LSR-PE system (Figure 7). To get a rank order of integration efficiency, we calculated the delta Ct by subtracting the Ct of the probes targeting the integration junction from the Ct of a reference genomic region. Integration efficiency varies by loci, LSR, length of attachment site, and linker (cis vs trans). ddPCR of donor integration at the ACTB and LMNB1 loci

Absolute integration efficiency was determined utilizing a single pegRNA by performing ddPCR of the integration junction and normalizing to an unedited locus (Figure 8A, 8B). All LSRs tested had detected LSR-mediated integration at the ACTB and LMNB1 locus, and no integration was seen in the PE-LSR-Donor and Donor only controls. Consistent with qPCR, trans delivery was slightly more efficient than cis delivery in all cases. qPCR of donor integration, 1 step delivery, 2 pegRNAs

Integration into the AAVS1 locus was detected across all LSRs, in both cis and trans (Figure 9 and Figure 10). The no donor control had undetected integration, and the donor only negative control had a Ct>35, which is above the threshold for reliable detection and is considered undetected. ddPCR of donor integration, 1 step delivery, 2 pegRNAs

Absolute integration efficiency of integration via 2 pegRNAs and LSR delivery in trans was determined by performing ddPCR of the integration junction and normalizing to an unedited locus. (Figure 10) LSRs integrated at an efficiency of 1-4%.

Example 2: Exemplary Sequences Example 3: Transgenic Animals

A system for stably integrating one or more nucleic acid sequences into a genome of a cell as provided herein is delivered to an embryonic stem cell of a non-human mammal (e.g., a mouse) to integrate a donor nucleic molecule containing a desired transgene into the genome of the embryonic stem cell.

In some cases, (a) a genome-editing system comprising (i) a polypeptide comprising a DNA binding domain and, optionally, a polymerase and (ii) a nucleic acid comprising a guide sequence that is complementary to a target site within said genome and a sequence that encodes an attA sequence; (b) a donor nucleic acid molecule comprising a transgene and an attD sequence; and (c) an integrase that targets said attA sequence and said attD site and can facilitate recombination between said attA site and said attD site are delivered to an embryonic stem cell of a non-human mammal (e.g., a mouse) to integrate the donor nucleic molecule containing the desired transgene into the genome of the embryonic stem cell.

The embryonic stem cell containing the transgene is injected into an inner cell mass of a blastocyst, and the blastocyst is then implanted into the uterus of female non-human mammal (e.g., a female mouse). Transgenic mice are selected from the offspring.

Example 4: Knock-out Animals

A system for stably integrating one or more nucleic acid sequences into a genome of a cell as provided herein is delivered to a non-human animal model (e.g., an adult mouse having a particular disease) to integrate a donor nucleic molecule containing a knock-out cassette into the genome of one or more cells within the non-human animal model.

In some cases, (a) a genome-editing system comprising (i) a polypeptide comprising a DNA binding domain and, optionally, a polymerase and (ii) a nucleic acid comprising a guide sequence that is complementary to a target site within said genome and a sequence that encodes an attA sequence; (b) a donor nucleic acid molecule comprising a knock-out cassette and an attD sequence; and (c) an integrase that targets said attA sequence and said attD site and can facilitate recombination between said attA site and said attD site are delivered to a non-human mammal (e.g., a mouse) to integrate the donor nucleic molecule containing the knock-out cassette into one or more cells within the non-human animal model.

Example 5: Generating engineered T cells

A system for stably integrating one or more nucleic acid sequences into a genome of a cell as provided herein is delivered to T cells to generate engineered T cells such as CAR T cells.

In some cases, (a) a genome-editing system comprising (i) a polypeptide comprising a DNA binding domain and, optionally, a polymerase and (ii) a nucleic acid comprising a guide sequence that is complementary to a target site within said genome and a sequence that encodes an attA sequence; (b) a donor nucleic acid molecule comprising a transgene encoding a particular receptor (e.g., a TCR or a CAR) and an attD sequence; and (c) an integrase that targets said attA sequence and said attD site and can facilitate recombination between said attA site and said attD site are delivered to T cells (e.g., T cells obtained from the mammal to be treated) to integrate the donor nucleic molecule containing the transgene encoding the particular receptor (e.g., the TCR or the CAR) into the T cells such that the particular receptor is expressed by the T cell (e.g., to generate an engineered T cell).

Example 6: Treating Cancer

A system for stably integrating one or more nucleic acid sequences into a genome of a cell as provided herein is delivered to T cells (e.g., T cells obtained from a mammal (e.g., a human) having cancer).

In some cases, (a) a genome-editing system comprising (i) a polypeptide comprising a DNA binding domain and, optionally, a polymerase and (ii) a nucleic acid comprising a guide sequence that is complementary to a target site within said genome and a sequence that encodes an attA sequence; (b) a donor nucleic acid molecule comprising a transgene encoding a receptor (e.g., a TCR or a CAR that can target an antigen expressed by cancer cells within a mammal) and an attD sequence; and (c) an integrase that targets said attA sequence and said attD site and can facilitate recombination between said attA site and said attD site are delivered to T cells (e.g., T cells obtained from the mammal to be treated) to integrate the donor nucleic molecule containing the transgene encoding the particular receptor (e.g., the TCR or the CAR) into the T cells such that the particular receptor is expressed by the T cell (e.g., to generate an engineered T cells).

The generated engineered T cells are administered to the mammal (e.g., a human) having cancer to treat the mammal.

Example 7: Treating Diseases Associated with Nucleotide Repeats

A system for stably integrating one or more nucleic acid sequences into a genome of a cell as provided herein is delivered to a mammal (e.g., a human) having a disease associated with nucleotide repeats (e.g., C9orf72 amyotrophic lateral sclerosis and frontotemporal dementia (C9 ALS/FTD)) to integrate a donor nucleic molecule containing a nucleic acid encoding a therapeutic gene product (e.g., a wild type C9orf72 polypeptide) to treat the mammal.

In some cases, (a) a genome-editing system comprising (i) a polypeptide comprising a DNA binding domain and, optionally, a polymerase and (ii) a nucleic acid comprising a guide sequence that is complementary to a target site upstream of a G4C2 repeat within said genome and a sequence that encodes an attA sequence; (b) a donor nucleic acid molecule comprising a splice acceptor, at least a portion of a wild type C9orf72 gene, and transcription termination signal and an attD sequence; and (c) an integrase that targets said attA sequence and said attD site and can facilitate recombination between said attA site and said attD site are delivered to cells within the mammal to integrate the donor nucleic molecule containing the splice acceptor, the at least a portion of a wild type C9orf72 gene, and the transcription termination signal into the cells such that a wild type C9orf72 polypeptide (e.g., a C9orf72 polypeptide lacking G4C2 hexanucleotide repeats associated with the C9 ALS/FTD) is expressed by the cells.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.