Related Applications

This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional application serial number 62/333580 filed May, 9, 2016, the disclosure of which is incorporated by reference herein in its entirety.

Field of the Invention

The invention relates, in part, to methods of designing and constructing gene drive systems and their inclusion and use in cell lines and organisms.

Background of the Invention

To date, gene drive elements based on Cas9 have been demonstrated in yeast (DiCarlo, J.E. et al., Nat Biotechnol. 2015 Dec;33(12): 1250-1255), fruit flies (Gantz, V. & Bier, E. 2015 Science 24 Apr: Vol. 348, Issue 6233, pp. 442-444), and two species of mosquitoes (Gantz, V. et al., 2015 PNAS Vol. 112 no. 49 E6736-E6743, doi:

10.1073/pnas.1521077112 , Hammond, A. et al., Nat Biotechnol. 2015 Dec 7;

doi: 10.1038/nbt.3439). Although functional gene drives have been prepared, gene drive systems are not available that are suitable for efficient and safe inclusion in wild populations of organisms. A type of gene drive, referred to as a "split drive" in which CRISPR gene drive components are separated such that cargo element A exhibits drive only in the presence of non-driving element B have been described (Esvelt, K, et al., 2014 eLife:e03401), demonstrated (DiCarlo, J.E. et al., Nat Biotechnol. 2015 Dec;33(12): 1250-1255), and recommended (Akbari, O. et al., Science 30 Jul 2015:DOI: 10.1126/science.aac7932) as a stringent laboratory confinement strategy that will prevent population alteration even if organisms accidentally escape. Although split gene drives provide a high level of confinement, they can be limited in their application. In addition, there are currently few options for controlling unauthorized or accidentally-released global drive systems.

Summary of the Invention

According to an aspect of the invention, methods constructing an evolutionarily stable gene drive system are provided, the methods including (a) selecting a gene drive system based on an RNA-guided DNA-binding protein nuclease; (b) creating a map of guide RNA for the selected gene drive system that denotes candidate sequence changes through the guide RNA structure, wherein the map is created based in part on sequence information of guide RNA in the selected gene drive system, variants thereof, and synthetic gene drive systems; (c) designing at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or more divergent guide RNA sequences each comprising one or more combinations of permutations of the candidate sequence changes independently selected to minimize the length of one or more sequences that are present in more than one guide RNA element sequence; (d) measuring a level of activity of the designed divergent guide RNA sequences; (e) identifying one or more of the designed divergent guide RNA sequences having a high level of the activity; and optionally, (f) including one or more of the identified guide RNA sequences in an evolutionarily stable gene drive system. In certain embodiments, the designed guide RNA sequences used reduce recombination within and/or between gene cassettes in the gene drive system more than would the use of identical guide RNA sequences yet are sufficiently active to enable gene drive. In certain embodiments, the identified guide RNA sequences comprise sequences with no more than 12, 13, 14, 15, 16, 17, 18, 19, or 20 base pairs in length homologous to any other. In some embodiments, the gene drive system includes elements comprising homologous sequences no more than 12, 13, 14, 15, 16, 17, 18, 19, or 20 base pairs in length. In some embodiments, the gene drive system is not a global RNA-guided drive system and has a statistically low probability of converting into a global RNA-guided gene drive system. In certain embodiments, a means of measuring the activity comprises a transcriptional activity reporter assay. In certain embodiments, a means of measuring the activity comprises a plasmid or phage exclusion assay. In some embodiments, the transcriptional activity reporter assay is the assay set forth in any embodiment of an aspect of the invention. In certain embodiments, the selected gene drive system is based on an RNA-guided DNA- binding protein endonuclease.

According to another aspect of the invention, methods of library-based RNA selection for guide RNAs suitable for construction of an evolutionarily stable gene drive system are provided, the methods including (a) selecting a gene drive system based on an RNA-guided DNA-binding protein nuclease; (b) creating a randomized library comprising a first plurality of candidate guide RNA sequences for the gene drive system wherein candidate guide RNA sequences include parent guide RNA sequences and variants thereof, wherein a guide RNA variant sequence comprises a parent guide RNA sequence with from one to five sequence mutations; (c) creating a targeted library comprising a second plurality of candidate guide RNA sequences for the gene drive system, wherein the candidate guide RNA sequences include parent guide RNA sequences and variants thereof, wherein a guide RNA variant sequence comprises a parent guide RNA sequence in which one or more base pairs in one or more predicted hairpin sequences are replaced with alternative base pairs that preserve the predicted hairpin structure (e.g. G-C pairs are replaced by C-G, A-T, T-A, G-T, and T-G) or that create a mispair (e.g. C-C); (d) measuring a level of an activity of the candidate guide RNA sequences from the created libraries; (e) identifying one or more of the candidate guide RNA sequences having a high level of the activity; and optionally, (f) including one or more of the identified guide RNA sequences in a gene drive system. In certain embodiments, a means of measuring in step (d) comprises: transforming or transfecting a bacterial or eukaryotic cell that expresses an active or inactive RNA-guided DNA nuclease enzyme, with one or more plasmids each comprising a candidate guide RNA sequence, a protospacer sequence targeted by a spacer in the candidate guide RNA, wherein the protospacer sequence is directly adjacent to the sequence encoding the candidate guide RNA such that an active candidate guide RNA cuts the plasmid; and detecting the cut plasmids as a measure of activity of the candidate guide RNA sequences, wherein the most active of the candidate guide RNAs are those most depleted when the RNA-guided DNA nuclease enzyme is active; and a means of identifying in step (e) comprises sequencing the plasmid sequences encoding the candidate guide RNA sequences before and after transformation. In some embodiments, the sequencing is high-throughput sequencing. In some embodiments, a means of measuring in step (d) comprises: transforming or transfecting a bacterial or eukaryotic cell that expresses an active or inactive RNA-guided DNA nuclease enzyme with one or more plasmids each comprising a candidate guide RNA sequence, two protospacer sequences targeted by a spacer in the candidate guide RNA, wherein an active candidate guide RNA cuts the plasmid at both protospacer sites, amplifying the region of the plasmid that contains both protospacer sequences and the candidate guide RNA sequence and size-select for the candidate guide RNAs that are missing the sequence between the protospacers, wherein the most active of the candidate guide RNAs are those for which the sequence between the protospacers is missing in the amplified regions; and a means of identifying in step (e) comprises sequencing the plasmid sequences encoding the candidate guide RNA sequences having a high level of the activity. In certain embodiments, a means of measuring the activity comprises a

transcriptional activity reporter assay, a plasmid exclusion assay or a phage exclusion assay. In certain embodiments, the transcriptional activation assay comprises use of a fluorescent reporter and fluorescence-assisted cell sorting to identify a level of guide RNA transcriptional activation. In certain embodiments, the identified guide RNA sequences comprise homologous sequences no more than 12, 13, 14, 15, 16, 17, 18, 19, or 20 base pairs in length. In some embodiments, the gene drive system includes elements comprising homologous sequences no more than 12, 13, 14, 15, 16, 17, 18, 19, or 20 base pairs in length. In some embodiments, wherein the RNA-guided DNA nuclease enzyme is Cas9, or a functional variant thereof. In certain embodiments, the RNA-guided DNA nuclease enzyme is Cpfl, or a functional variant thereof. In certain embodiments, the selected gene drive system is a CRISPR gene drive system. In some embodiments, the method also includes determining a base substitution in a hairpin in a candidate guide RNA variant that does not eliminate or significantly reduce an activity of its parent candidate guide RNA. In some embodiments, the method also includes determining a base mutation in a candidate guide RNA variant that does not eliminate or significantly reduce an activity compared to the activity of its parent candidate guide RNA. In certain embodiments, the method also includes determining one or more of a base insertion or a base deletion in a candidate guide RNA variant that does not eliminate or significantly reduce an activity compared to the activity of its parent candidate guide RNA.

According to another aspect of the invention, methods of measuring guide RNA activity via a transcriptional activation reporter using dCas9-VPR, are provided, the methods including (a) growing mammalian cells; (b) transfecting the mammalian cells with plasmids encoding: i) dCas9-VPR or the equivalent dead-nuclease transcriptional activator variant of the RNA-guided DNA-binding protein nuclease matching the guide RNAs to be tested, ii) the guide RNA to be evaluated, iii) a reporter plasmid comprising a minimal promoter and one or more protospacer binding sites upstream of a gene encoding a fluorescent protein, and iv) a control plasmid expressing a different fluorescent marker gene as a transfection control marker; and (c) analyzing the transfected cells by flow cytometry and measuring activity, ignoring any that do not fluorescent due to the transfection control marker. In some embodiments, the cells were HEK293T cells and were grown in Dulbecco's Modified Eagle Medium fortified with 10% FBS and Penicillin/Streptomycin and were incubated at a constant temperature of 37°C with 5% C0 ₂ . In some embodiments, prior to transfection, the grown cells are split cells into 24-well plates, divided into approximately 50,000 cells per well. In certain embodiments, the means of transfection comprises: using 2μ1 of

Lipofectamine 2000 with 200ng of dCas9 activator plasmid, 25ng of guide RNA plasmid, 60ng of reporter plasmid and 25ng of EBFP2 expressing plasmid, wherein the reporter plasmid is a modified version of addgene plasmid #47320, a reporter expressing a tdTomato fluorescent protein adapted to contain an additional gRNA binding site lOObp upstream of the original site, and the activator is a tripartite transcriptional activator fused to the C-terminus of nuclease-null Streptococcus pyogenes Cas9. In some embodiments, if a library of guide RNAs is being simultaneously analyzed, a means of analyzing in step (c) comprises using use fluorescent-assisted cell sorting (FACS), to sort for plasmids encoding highly active guide RNAs and sequencing to identify the sequences.

According to another aspect of the invention, methods for constructing an

evolutionarily stable gene drive system, are provided, the methods including: (a) selecting a gene drive system based on an RNA-guided DNA-binding protein nuclease; (b) selecting a first gene to be targeted that is either haploinsufficient for normal cell growth, or is a gene encoding a ribosomal protein; and (c) selecting a second gene that when initially expressed in an organism of interest, is only expressed in the organism's germline after soma-germline differentiation in the subject, and wherein the second gene's promoter/enhancer/3'UTR combined with a coding DNA sequence transgene encoding an RNA-guided DNA-binding protein nuclease lead to similar tissue-and temporally-specific expression. In some embodiments, the haploinsufficient gene is a gene for which a single copy is insufficient for normal growth and division of a cell. In certain embodiments, a means for selecting the second gene comprises detecting timing of the second gene's expression in the organism. In some embodiments, a means for detecting time comprises an expression assay of any embodiment of one of an aspect of the invention. In some embodiments, the gene drive cassette further comprises a sequence encoding one or more guide RNA sequences that target the first gene's wild-type locus. In some embodiments, the guide RNA sequences are expressed from a promoter that is not a promoter of the second gene. In certain

embodiments, a means for selecting the promoter comprises a method of any embodiment of one of an aspect of the invention. In some embodiments, the DNA-binding protein nuclease is attached (fused) to a detectable label. In some embodiments, the detectable label is a fluorescent label. In certain embodiments, the method also includes: (d) expressing in a cell, a gene drive cassette comprising: the second gene's promoter/enhancer/3'UTR sequence, the RNA-guided DNA binding protein nuclease sequence; and at least one guide RNA sequence. In some embodiments, the cell is in an organism. In some embodiments, the organism is an embryonic organism. In some embodiments, the method also includes detecting the detectable label in the embryonic organism and verifying that the expression of the first gene is germline-specific and occurs at a predetermined developmental stage in the embryonic organism. In certain embodiments, the method also includes crossing the organism with a wild-type of the organism and comparing the fertility of the organism with a control fertility. In some embodiments, the control fertility is the fertility of a cross between two of the wild- type organisms, neither of which includes a cell comprising the gene drive cassette. In some embodiments, the method also includes assaying an offspring of the organism to detect a heterozygote in which the gene drive is not present. In certain embodiments, the selected gene drive system is a gene drive system based on an RNA-guided DNA-binding protein nuclease.

According to another aspect of the invention, methods of assaying a candidate gene for haploinsufficiency in the germline of an organism are provided, the methods including:

(a) selecting a gene drive system based on an RNA-guided DNA-binding protein nuclease;

(b) creating a first strain of transgenic organisms in which an RNA-guided DNA-binding protein nuclease is expressed exclusively in the germline after soma-germline differentiation;

(c) preparing a second strain of transgenic organisms in which a single guide RNA targeting the coding region of a candidate haploinsufficient gene is expressed under a polymerase III promoter; and wherein the target sites of the candidate haploinsufficient gene are mutated to prevent cutting; (d) crossing the first strain and second strain of transgenic organisms to create a heterozygous hybrid organism strain in which the wild-type copy of the candidate haploinsufficient gene is predicted to be cut in germline cells in the hybrid organism strain just after soma-germline differentiation in the hybrid organism strain; and (e) harvesting mature gametes from the hybrid organism and sequencing to measure the fraction that contain copies of the target gene that were mutated to avoid cutting rather than copies inactivated by cutting; wherein a high fraction corresponds to a gene that is haploinsufficient in the germline. In some embodiments, the method also includes: (f) expressing in an organism, a gene drive system in which a target gene of the gene drive is the candidate gene identified in step (b) as haploinsufficient in the germline of the organism. In some embodiments, the selected gene drive system is a gene drive system based on an RNA-guided DNA-binding protein nuclease. In certain embodiments, the means of selecting the promoter in step (c) comprises a method of any embodiment of one of an aspect of the invention set forth herein.

According to another aspect of the invention, methods for assessing timing of expression of a gene drive cassette in a cell in an embryonic organism are provided, the methods including (a) expressing in a cell in an embryonic organism, a gene drive cassette comprising: a promoter/enhancer/3'UTR sequence, an RNA-guided DNA binding protein nuclease gene sequence, and one or more RNA guide sequences, wherein: (i.) when expressed in the cell: the promoter/enhancer/3'UTR drives expression of the RNA-guided DNA binding protein nuclease gene, and the RNA guide sequences target a predetermined haploinsufficient gene; (b) isolating a germline cell from the embryonic organism; (c) determining expression of the promoter/enhancer/3'UTR in the isolated germline cell; and (d) identifying whether the gene drive cassette is only expressed in the germline after the soma- germline differentiation in the embryonic organism. In some embodiments, In some embodiments, the means of determining in step (c) comprises performing a full transcriptome sequencing analysis of the germline cell. In some embodiments, the means of determining in step (c) comprises testing the promoter/enhancer/3'UTR for appropriate expression in the germline cell. In certain embodiments, the method also includes, (e) encoding a fluorescent marker gene, such as green fluorescent protein, adjacent to the RNA-guided DNA-binding protein nuclease such that the two are transcriptionally and translationally coupled, as can be achieved through the use of a 2A peptide fusion, and visualizing the expression pattern of the fluorescent marker in relevant tissues of the organism. In some embodiments, the method also includes, (f) expressing in an organism, a gene drive comprising the gene drive cassette identified in step (d) as only expressed in the germline after soma-germline differentiation.

According to another aspect of the invention, methods of determining an activity level of a candidate promoter for guide RNA expression in a gene drive system are provided, the methods including: (a) expressing in an organism or cell line that expresses an RNA-guided DNA nuclease, a candidate promoter sequence that drives a guide RNA when expressed in the organism or cell line, wherein the guide RNA targets one or two sequences either upstream or downstream of the candidate promoter sequence; (b) isolating DNA from the organism or cell line of (a) in which the candidate promoter sequence has been expressed; (c) amplifying the (i.) one or more sequences targeted by the guide RNA and (ii.) the candidate promoter sequence in the isolated DNA, forming amplicons; (d) identifying the amplicons of the targeted sequences and the candidate promoter sequence; (e) determining a level of activity of the candidate promoter sequence, based on the abundance of the identified amplicons of step (d); (f) sequencing the amplicons to identify the sequence of a candidate promoter determined to have the high level of activity; and optionally (g) selecting the candidate promoter determined to have the high level of activity and including the selected candidate promoter in a gene drive system. In some embodiments, the means for preparing amplicons comprises polymerase chain reaction methods. In certain embodiments, when the guide RNA targets two target site sequences, the identification in step (d) of an amplicon of the two target sites that does not include the sequence between the two target sites, indicates a high level of activity of the candidate promoter sequence. In some embodiments, the gene drive system is based on an RNA-guided DNA-binding protein nuclease. According to another aspect of the invention, methods of determining activity of a candidate promoter for guide RNA expression in a gene drive system are provided, the methods including: (a) expressing a DNA molecule in cells of an organism or cell line that express an RNA-guide DNA nuclease, wherein: (i.) sequences encoded by the DNA molecule comprise: a guide RNA sequence; a candidate promoter sequence that drives expression of the guide RNA in the cell; a first fluorescent protein; and a second fluorescent protein; (ii.) the guide RNA targets two sequences positioned in the DNA molecule such that expression of the first fluorescent protein is disrupted by activity of the promoter; and (b) identifying a level of activity of a candidate promoter when the DNA is expressed in the cells. In certain embodiments, a means for identifying in step (b) comprises fluorescence-assisted cell sorting (FACS) to enrich for cells comprising the second fluorescent protein but not the first fluorescent protein, wherein a higher level of the second fluorescent protein compared to a level of the first fluorescent protein in one or more cells expressing the DNA, identifies a candidate promoter in the one or more cells as having a high level of activity. In some embodiments, the method also includes sequencing the DNA of an identified high-activity candidate promoter. In some embodiments, the method also includes selecting a candidate promoter identified as having a high level of activity, and including the selected candidate promoter in a gene drive system. In some embodiments, the gene drive system is based on an RNA-guided DNA-binding protein nuclease.

According to another aspect of the invention, methods of designing a daisy chain gene drive system are provided, the methods including: (a) selecting a daisy chain gene drive for inclusion in a first organism, wherein the daisy chain gene drive comprises N elements, where N is a total number of elements in the daisy chain, and each element except one comprises one or more sequence-divergent guide RNA sequences; (b) designing an additional effector element that encodes one or more guide RNAs and when present in a genome of the first organism comprising the N-element daisy chain gene drive of step (a): the additional element exhibits drive in the presence of the appropriate RNA-guided DNA nuclease wherein: (i.) including the additional element the genome of the first organism comprising the N-element daisy chain gene drive results in a complete N+effector-element daisy drive organism; (ii.) including the additional element in a second organism of the same species as the first organism but without the N-l daisy chain gene drive, and crossing the first organism comprising the N-l daisy chain gene drive and the second organism results in one or more offspring comprising an N-element daisy chain gene drive wherein the additional element drives in the presence of the RNA-guided DNA nuclease and alters or suppresses the activity of the N-l daisy chain gene drive; and (iii.) releasing a plurality of the first organism comprising the N-element daisy chain gene drive into an environment initiates an N-element daisy chain gene drive effect that spreads the gene encoding the RNA-guided DNA nuclease through a local population of an organism of the same species, wherein releasing one, two, or a plurality of a third organism of the same species, each comprising a genome encoding one or more of an independently selected modulating additional effector element that when expressed in an organism comprising the N-element daisy chain gene drive results in a modulating effect in the local population of organisms. In certain embodiments, suppressing the N-element daisy drive effect removes the RNA-guided DNA nuclease from the local population. In some embodiments, releasing the one, two, or the plurality of the third organisms, each comprising one or more independently selected modulating effector elements into the population in series or in parallel, alters the population impacted by the N- element-daisy drive effect two or more times. In some embodiments, the method also includes preparing the designed daisy chain gene drive system. In some embodiments, the daisy chain gene drive system is a daisy chain gene drive system based on an RNA-guided DNA-binding protein nuclease.

According to another aspect of the invention, methods of preparing an N-l daisy chain gene drive organism are provided, the methods including: (a) selecting a daisy chain gene drive system; (b) identifying a plurality of sequence-divergent guide RNAs for inclusion in a desired number (N-l) of daisy chain drive elements; and (c) identifying N target genes, an RNA-guided DNA nuclease, and expression conditions suitable for expressing the one or more RNA-guided DNA nucleases. In some embodiments, all but one of the daisy chain gene drive elements requires two or more guide RNAs. In certain embodiments, the method also includes minimizing a potential for recombination between two or more elements of the N-l daisy chain by maximizing the sequence-divergence of the daisy chain gene drive elements. In some embodiments, the method also includes defining the positions of the elements the daisy chain gene drive in descending order. In some embodiments, the element order is assigned alphabetical indicators and position A is the position of the desired modulation element, wherein preparing a multi -element daisy chain gene drive defined as having element positions .. E-D-C-B-A-effector, comprises creating a gene drive comprising elements .. E-D-C-B-A to which the desired modulation effector element can be added in position "effector", wherein each of the elements comprises an independently selected gene drive cassette comprising: a sequence encoding a promoter and one or more RNA guide sequences; and preparing the daisy chain gene drive comprises: (d) selecting a target gene of the element A gene drive cassette; (e) recoding the element A target gene; (f) encoding an RNA-guided DNA nuclease downstream of the 3'UTR in the element A gene drive cassette; (g) selecting a target gene of the element B gene drive cassette; (h) designing the element B gene drive cassette construct, wherein the cassette encodes one or more guide RNAs that recognize the selected target gene of the A element and are positioned downstream of the B element target gene; (i) recoding the element B target gene and replacing the element B target gene's 3'UTR with a 3'UTR from another gene with similar expression conditions; (j) repeating steps (h)-(i) for each of the higher elements (D, E, and above) until all the desired drive elements have been prepared, optionally encoding and therefore relying on one or more different RNA-guided DNA-binding protein nucleases for different elements in the chain; and (k) combining all of the elements except the modulating element in a single organism or cell line strain. In some embodiments, the element order is assigned sequential indicators and position A is the position of the desired nuclease-encoding element, wherein preparing a multi-element daisy chain gene drive defined as having element positions .. E-D-C-B-A- effector, comprises creating a gene drive comprising elements .. E-D-C-B-A to which the desired modulation effector element can be added in position "effector", wherein each of the elements comprises an independently selected gene drive cassette comprising: a sequence encoding a promoter and one or more RNA guide sequences; and preparing the daisy chain gene drive comprises: (d) selecting a target gene of the element A gene drive cassette; (e) recoding the element A target gene; (f) encoding an RNA-guided DNA nuclease downstream of the 3'UTR in the element A gene drive cassette; (g) selecting a target gene of the element B gene drive cassette, wherein the element B is in a neutral site; (h) designing the element B gene drive cassette construct, wherein the cassette encodes one or more guide RNAs that recognize the selected target gene of the A element and are positioned downstream of the B element target gene; (i) not recoding the element B target gene; (j) repeating steps (h)-(i) for each of the higher elements (C, D, E, and above) until all the desired drive elements have been prepared, optionally encoding and therefore relying on one or more different RNA- guided DNA-binding protein nucleases for different elements in the chain; and (k) combining all of the elements except the modulating element in a single organism or cell line strain. In certain embodiments, the element order is assigned sequential indicators and position A is the position of the desired nuclease-encoding element, wherein preparing a multi -element daisy chain gene drive defined as having element in positions .. E-D-C-B-A-effector, comprises creating a gene drive comprising elements .. E-D-C-B-A to which the desired modulation effector element can be added in position "effector", wherein each of the elements comprises an independently selected gene drive cassette comprising: a sequence encoding a promoter and one or more RNA guide sequences; and preparing the daisy chain gene drive comprises: (d) selecting a target gene of the element A gene drive cassette, wherein the element A target gene is in a neutral site; (e) not recoding the element A target gene; (f) encoding an RNA- guided DNA nuclease downstream of the 3'UTR in the element A gene drive cassette; (g) selecting a target gene of the element B gene drive cassette, wherein the element B target gene is in a neutral site; (h) designing the element B gene drive cassette construct, wherein the cassette encodes one or more guide RNAs that recognize the selected target gene of the A element and are positioned downstream of the B element target gene; (i) not recoding the element B target gene; (j) repeating steps (h)-(i) for each of the higher elements (C, D, E, and above) until all the desired drive elements have been prepared, wherein each element encodes guide RNAs that target the wild-type locus of the next in the chain as well as the A element, optionally encoding and therefore relying on one or more different RNA-guided DNA- binding protein nucleases for different elements in the chain; and (k) combining all of the elements except the modulating element in a single organism or cell line strain. In some embodiments, the recoded A element gene is fused to a 2A peptide and the nuclease gene. In certain embodiments, the recoded "A" element in mammals comprises (recoded Nanos3 last exon)-(2A)-(Cpfl)-(Nanos3UTR). In some embodiments, a means of combining all of the elements except for the modulating element in a single organism strain comprises crossing organisms comprising one or more of the daisy chain gene drive elements other than the modulating element, wherein the resulting organism strain when released into a local population of the organism strain not comprising the daisy chain gene drive, will exhibit daisy chain gene drive to spread element B, which encodes the RNA-guiding DNA-binding nuclease, through the local population. In some embodiments, a means of recoding the element A target gene in step (e) comprises a method of any embodiment of an aspect of the invention disclosed herein. In certain embodiments, a means of recoding the element B target gene and replacing the element B target gene's 3'UTR replaced with a 3'UTR from another gene with similar expression conditions in step (i) comprises a method of any embodiment of an aspect of the invention disclosed herein. In some embodiments, for an application comprising population suppression using a sex-specific effect, the highest/proximal element in the daisy chain (e.g. E in an E-D-C-B-A chain) is encoded within a locus exclusive to the unaffected sex. In some embodiments, the method also includes preparing a modulating organism strain comprising the daisy chain gene drive modulating effector element, wherein the modulating element targets the target gene selected in step (c) and comprises a DNA sequence encoding the desired modulating genomic change and guide RNAs capable of driving the genomic change by cutting the target gene of the modulating element. In some embodiments, the method also includes releasing a plurality of organisms of the strain prepared in step (k) into a local population of the wild-type strain. In certain embodiments, the method also includes releasing a plurality of the modulating organism strain into the local population. In some embodiments, a means for identifying plurality of sequence-divergent guide RNAs comprises a method of any embodiment of one of the aforementioned aspects of the invention.

According to another aspect of the invention, methods of selecting a promoter for expression of multiple guide RNAs with minimal homology in a gene drive system are provided, the methods including: (a) selecting a gene drive system; and (b) identifying one or both of: (i.) at least one polymerase III promoter capable of strong RNAi or guide RNA expression and suitable for use in the gene drive system; and (ii.) at least one promoter suitable for use in the gene drive system. In some embodiments, a first and a second guide RNA may both be expressed from the same polymerase III promoter, wherein the gene drive cassette comprising the promoter and guide RNA sequences in which a poly-T stretch leading to transcriptional termination of the first guide RNA is replaced with a 10-15 base pair linker to the second guide RNA. In certain embodiments, an identified promoter is suitable for use in the gene drive system and expresses two or more guide RNAs. In some embodiments, a means of expressing the two or more guide RNAs from the single promoter, comprises a tRNA-based processing strategy. In some embodiments, the gene drive system comprises a CRISPR system that does not require an external processing factor for its arrays. In certain embodiments, the CRISPR system comprises a CRISPR/Cpfl system. In some

embodiments, the polymerase III promoter is U6, HI, or a tRNA promoter. In some embodiments, the identified promoter is a polymerase II promoter. In some embodiments, the promoters are identified using a method of any embodiment of any of the aforementioned aspects of the invention.

According to another aspect of the invention, methods of identifying the efficacy of a tRNA in tRNA-guide RNA-tRNA array processing are provided, the methods including: (a) selecting a gene drive system; (b) preparing an organism strain comprising the gene drive system in which an RNA-guided DNA nuclease is expressed using a housekeeping gene enhancer/promoter/3'UTR that also expresses a fluorescent protein, either from a separate promoter or via a 2A peptide fusion; (c) preparing one or more additional strains of the organism comprising the gene drive system in which a promoter predetermined to be effective in the selected gene drive is expressed in one or more cells of the organism in which the promoter drives a construct consisting of a tRNA, a control guide RNA that does not target any sequence in the cell, a different tRNA to be tested, a guide RNA targeting the gene encoding the fluorescent protein or an equivalent recessive marker gene, a third tRNA, and another control guide RNA; (d) crossing the strain of (b) with a strain of (c); (e) measuring the fluorescence in the progeny of the cross to identify the efficacy of the tRNA in the processing, wherein the level of the fluorescence corresponds inversely to the efficacy of the tRNA processing; and (f) repeating steps (b) - (e) varying the different tRNAs in the second and third positions, until sufficient tRNAs have been identified for processing a desired number of daisy drive elements of the selected gene drive. In certain embodiments, the housekeeping gene is an actin gene. In some embodiments, the promoter is identified according to a method of any embodiment of one of the aforementioned aspects of the invention.

According to another aspect of the invention, methods of identifying the efficacy of a candidate tRNA in tRNA-guide RNA-tRNA array processing are provided, the methods including: (a) selecting a gene drive system; and (b) designing a cell culture strain of the selected gene drive system, wherein the cell culture strain cells comprise: a DNA construct: (i.) sequences encoding a first fluorescent protein and an RNA-guided DNA nuclease, and a housekeeping gene enhancer/prom oter/3'UTR sequence that drives expression of the sequences of (i.); (ii.) sequences encoding a tRNA, a control guide RNA that does not target any sequence in the cell, a candidate tRNA to be tested for efficacy, a guide RNA targeting the gene encoding the first fluorescent protein or an equivalent recessive marker gene, a third tRNA, another control guide RNA, and a promoter that drives expression of the sequences of (ii.); and (iii.) a sequence encoding a second fluorescent protein. In some embodiments, the method also includes, (c) preparing the designed cell culture strain and (d) identifying efficacy of the tRNA in the designed cell culture strain. In certain embodiments, a means for identifying in step (d) comprises fluorescence-assisted cell sorting (FACS) to enrich for cells of the cell culture strain comprising the second fluorescent protein but not the first fluorescent protein, wherein a higher level of the second fluorescent protein compared to a level of the first fluorescent protein in one or more cells of the cell culture strain expressing the DNA, identifies the candidate tRNA as efficacious in the one or more cells. In some embodiments, the method also includes sequencing the DNA of the efficacious tRNA. In some

embodiments, the method also includes selecting a candidate tRNA identified as efficacious, and including the selected candidate tRNA in a gene drive system. In certain embodiments, the gene drive system is a CRISPR gene drive system.

According to another aspect of the invention, methods of identifying the efficacy of candidate tRNAs in tRNA-guide RNA-tRNA array processing are provided, the methods including: (a) selecting a gene drive system; (b) preparing a library of DNA fragments encoding: (promoter-(sitel)-(sitel)-tRNAl-(guide RNA targeting site l)-tRNA2-(guide RNA targeting site 2)-tRN A3 -(control guide RNA)-(site 2)-(site2) for many different tRNAs of interest in different combinations, wherein tRNAl, tRNA2, and tRNA3 are candidate tRNAs; (c) delivering the DNA fragments from the prepared library into cells of a target organism species under conditions in which the appropriate RNA-guided DNA-binding protein nuclease is also produced; (d) extracting DNA from the cells; (e) amplifying the extracted DNA using flanking primers to amplify the region encompassing sites 1 and sites 2 and the region between them to produce amplicons; and (f) sequencing the amplicons, wherein the presence of sequences that include mutations in site 1 or site 2 indicates efficacy of the candidate tRNAs. In some embodiments, the method also includes selecting one or more of the efficacious candidate tRNAs, and including the selected candidate tRNAs in a gene drive system. In some embodiments, the gene drive system is based on an RNA-guided DNA- binding protein nuclease.

According to another aspect of the invention, methods of recoding a target gene are provided, the methods including: (a) selecting a gene drive system; (b) identifying a target site with few off-targets within a coding sequence of a first gene for inclusion in the gene drive system; (c) recoding the coding sequence of the first gene from the target site to the end of the coding sequence by changing at least every fourth codon if possible, removing all introns in between, and replacing the gene's 3'UTR with a 3'UTR from a second gene with similar expression conditions to those of the first gene; and (d) encoding one or both of: one or more guide RNAs and/or an RNA-guided DNA nuclease downstream of the 3'UTR, wherein there is no homology to the wild-type DNA sequence between the new 3'UTR and any of the encoded elements. In some embodiments, a means of encoding one or more guide RNAs in step (d) includes a method of any one embodiment of any of the aforementioned aspects of the invention. In some embodiments, the method also includes, including the recoded target gene in a gene drive system. In certain embodiments, the gene drive system is based on one or more RNA-guided DNA-binding protein nucleases.

According to another aspect of the invention, methods of suppressing an organism population via genetic load are provided, the methods including: (a) selecting a gene drive system and a daisy chain gene drive organism strain comprising one or more of each of: a target gene, an N-element daisy chain gene drive, a gene drive cassette comprising a promoter/enhancer/3'UTR sequence, an RNA-guided DNA-binding protein nuclease sequence, and one or more guide RNA sequences; (b) identifying one or more recessive target gene(s) that are not included in the N-element daisy chain gene drive of step (a) and that correspond to: sex-specific infertility, infertility, sex-specific viability, or viability of an organism species; (c) creating: (i.) a first effector strain of a cell or organism in a wild-type background of the organism species for one of the identified genes, wherein a large portion or all of the identified gene is replaced by one or more guide RNAs that target the wild-type identified gene, and exhibit drive in the presence of a RNA-guided DNA nuclease in the gene drive system; or (ii.) a second effector strain of a cell or organism comprising a daisy drive element via genetic recoding of the identified gene, wherein the daisy drive element expresses two guide RNAs and is capable of drive by targeting a wild-type version of the identified gene in the presence of an RNA-guided DNA nuclease and the two guide RNAs, and the drive disrupts the identified gene; (d) crossing the first created effector strain or the second created effector strain with an N-element daisy drive strain creating a complete daisy drive strain; (e) optionally homozygosing the complete daisy drive strain; and optionally (f) inhibiting a suppression activity in the complete daisy drive strain _a wherein if the suppression method is sex-specific, a proximal element of the daisy drive strain of (e) is located within a locus exclusive to the unaffected sex. In some embodiments, a created strain is an organism. In some embodiments, a means of creating a second strain of a cell or organism comprising a daisy drive element in step (c)(ii.) comprises a method of any embodiment of one of the aforementioned aspects of the invention. In some embodiments, a means of creating the N- element daisy drive strain of step (d) comprises a method of any embodiment of one of the aforementioned aspects of the invention. In certain embodiments, a means of inhibiting a suppression activity in the complete daisy drive strain comprises a method of any

embodiment of one of the aforementioned aspects of the invention. In some embodiments, a means of inhibiting a suppression activity in the complete daisy drive strain comprises a method comprising a CRISPR nuclease inhibitor, wherein the CRISPR nuclease inhibitor blocks cutting. In some embodiments, the CRISPR nuclease inhibitor inhibits suppression and permits maintenance of the complete daisy drive strain in a background that encodes the CRISPR nuclease inhibitor. In certain embodiments, the method also includes: (g) determining a number of organisms of the created organism strain that must be added into a pre-determined size of a wild-type target population to suppress the target population to a desired size. In some embodiments, a means for the determining comprises preforming cage studies and field trials. In some embodiments, a means for the determining comprises:

sampling the target wild-type population and estimating the number of organisms required for release. In some embodiments, the method also includes: (h) releasing a number of the daisy drive organism into a local environment of the target wild-type population effective to suppress or eliminate the target wild-type population from the local environment. In certain embodiments, the gene drive system is based on one or more RNA-guided DNA-binding protein nucleases.

According to another aspect of the invention, methods of suppressing a population of an organism by causing a gene drive-carrying organisms to develop as a single sex are provided, the methods including: (a) selecting a gene drive system and selecting an evolutionarily stable daisy chain gene drive organism strain comprising one or more of each of: a target gene, an N-element daisy chain gene drive, a gene drive cassette comprising a promoter/enhancer/3'UTR sequence, an RNA-guided DNA-binding protein nuclease sequence, and one or more guide RNA sequences; (b) identifying a target gene that is not included in the N-element daisy chain gene drive of step (a) and whose presence or disruption causes an organism to develop as a particular sex; (c) creating (i.) a first transgenic organism strain comprising a new effector daisy drive element for the gene drive system, via genetic recoding wherein the organism expresses one or more guide RNAs that will allow the new element to exhibit drive by targeting the wild-type version of the identified gene in the presence of an RNA-guided DNA nuclease and the guide RNA(s), wherein the inclusion of the new daisy drive element results in organisms of the first transgenic organism strain to develop as the particular sex; or (ii.) a second transgenic organism strain with a new daisy drive element for the gene drive system comprising a recoded identified gene, wherein the new daisy drive element expresses one or more guide RNAs and is capable of drive by targeting the wild-type version of the identified gene in the presence of an RNA-guided DNA nuclease and the one or more guide RNAs, and the new daisy drive element cause the organism to develop as the particular sex. (d) crossing an organism of the first transgenic organism strain or the second transgenic strain with an N-element daisy drive organism creating a complete daisy drive strain; (e) homozygosing the complete daisy drive strain; and (f) determining a number of the complete daisy drive strain organisms that must be added into a pre-determined size of a population of a wild-type strain of the daisy drive strain organisms to suppress the population of the wild-type strain to a desired size. In some embodiments, the target gene in step (b) is identified using standard genetic methods. In some embodiments, a means for creating the first transgenic organism strain comprising a new daisy drive element for the gene drive system in step (c)(i) comprises a method of any embodiment of any aspect of the invention set forth herein. In certain embodiments, a means for creating the second transgenic organism strain with a new daisy drive element for the gene drive system in step (c)(ii) comprises a method of any embodiment of any aspect of the invention set forth herein. In some embodiments, a means creating the strain with an N- element daisy drive of step (d) comprises a method of any embodiment of any aspect of the invention set forth herein. In some embodiments, the target gene is Sry and is identified at least in part because it causes maleness m Mus musculus, the target gene Nix is identified at least in part because it causes maleness in Aedes aeg pti, and the target gene fem-3 is identified because its loss causes femaleness in C. elegans. In some embodiments, a means for the determining comprises preforming cage studies and field trials. In certain

embodiments, a means for the determining comprises: sampling the target wild-type population and estimating the number of organisms required for release. In some

embodiments, the gene drive system is a CRISPR gene drive system.

According to another aspect of the invention, methods of suppressing a population via chromosomal shredding are provided, the methods including: (a) selecting a gene drive system; (b) determining a sex against which to bias a population of organisms; (c) identifying a set of sequences on either side of the centromere of a sex chromosome corresponding to the sex determined in step (b); (d) selecting an evolutionarily stable daisy chain gene drive organism strain comprising one or more of each of: a target gene, an N-element daisy chain gene drive, a gene drive cassette comprising a promoter/enhancer/3'UTR sequence, an RNA- guided DNA-binding protein nuclease sequence, and one or more guide RNA sequences; (e) determining a target gene that is not included in a N-element daisy chain gene drive (e.g. D- C-B) background in the evolutionarily stable daisy chain gene drive system; (f) recoding the determined target gene; (g) encoding in a new daisy drive effector element: (1) at least one guide RNA that targets the wild-type version of the target gene that function with the RNA- guided DNA nuclease encoded in a gene-drive element of the evolutionarily stable daisy drive strain other than the new gene-drive element; and (2) in separate strains, one of: (i.) an orthogonal RNA-guided DNA nuclease encoded such that it is expressed exclusively during late meiosis; or (ii.) one or more guide RNAs for the orthogonal RNA-guided DNA nuclease that target the sequences identified in step (c); (h) preparing an organism strain comprising the encoded daisy chain gene drive element of step (g) and the evolutionarily stable daisy chain gene drive selected in step (d); (i) determining a number of the offspring that must be added into a pre-determined-size population of the organism strain that does not include the evolutionarily stable daisy chain gene drive, to effectively suppress the population to a desired size, and optionally (j) releasing the determined number of daisy chain gene drive organisms prepared in step (h) into the population of the organism strain that does not include the evolutionarily stable daisy chain gene drive. In some embodiments, a means for determining a target gene that is not included in a N-element daisy chain gene drive (e.g. C- B-A) background in the evolutionarily stable daisy chain gene drive system in step (e) comprises a method of any embodiment of any aforementioned aspect of the invention. In certain embodiments, a means for recoding the determined target gene in step (f) comprises a method of any embodiment of any aspect of the invention set forth herein. In some embodiments, a means for preparing the organism strain of step (h) comprises one of: (i.) crossing the prepared organism strain of (h) with non-favored-sex members of the wild-type organism strain, wherein the offspring produced in the cross are daisy chain gene drive containing organisms suitable for release to suppress a wild-type population size; or (ii.) generating a strain of the organism comprising only the proximal element of the daisy chain gene drive system (e.g. element D for a D-C-B-A system) and crossing the prepared organism strain of (h) with the generated strain that is sorted for the non-favored sex; wherein the organisms produced in cross (i.) or cross (ii.) are daisy chain gene drive containing organisms suitable for release. In some embodiments, a means in step (i.) for crossing the prepared organism strain of (h) with non-favored-sex members of the wild-type organism strain, wherein the offspring produced in the cross are daisy chain gene drive containing organisms suitable for release to suppress a wild-type population size; comprises a method of any embodiment of any aspect of the invention set forth herein. In some embodiments, a means for the determining in step (i) comprises preforming one or more of: a cage study and a field trial. In certain embodiments, a means for the determining in step (i) comprises: sampling the target wild-type population and estimating the number of organisms required for release. In some embodiments, the gene drive system is based on one or more RNA-guided DNA- binding protein nucleases.

According to another aspect of the invention, methods of suppressing an organism population are provided, the methods including: (a) identifying an organism population of interest to suppress; (b) selecting a gene drive system; (c) identifying one or more recessive target genes corresponding to, in order of preference, sex-specific infertility, infertility, sex- specific viability, or viability; (d) preparing a first strain of an organism of the identified organism population that encodes a generic daisy chain gene drive comprising one or more of each of: a target gene, an N-element daisy drive, a gene drive cassette comprising a promoter/enhancer/3'UTR sequence, an RNA-guided DNA-binding protein nuclease sequence, and one or more guide RNA sequences; (e) preparing a second strain of an organism of the identified organism population that encodes one or more daisy effectors wherein each identified target gene sequence is replaced with one or more guide RNAs that target sequences within the identified wild-type recessive gene sequences; (f) determining a number of the prepared daisy chain gene drive strain organisms of (d) that must be added into a pre-determined-size population of the organism strain that does not include the daisy chain gene drive to effectively spread the RNA-guided nuclease through the pre-determined-size population and prepare the pre-determined-size population for suppression, and optionally (g) releasing the determined number of the prepared daisy chain gene drive strain organisms into the population of the organism strain that does not include the daisy chain gene drive, wherein the release prepares the organism population for subsequent alteration or suppression, and optionally (h) releasing a plurality of organisms of the second strain wherein the release suppresses the identified organism population. In some embodiments, a means for encoding the guide RNAs in step (c) comprises a method of any embodiment of any aspect of the invention set forth herein. In certain embodiments, the daisy chain gene drive system is based on one or more RNA-guided DNA-binding protein nucleases. In some embodiments, the spread of the generic daisy drive organism is monitored prior to release of organisms carrying effector elements. In some embodiments, additional daisy drive or wild-type organisms are released to adjust the scope of the population to be suppressed prior to releasing organisms of the second strain.

According to another aspect of the invention, methods of suppressing the number of organisms in a population by biasing toward one sex are provided, the methods including: (a) selecting a gene drive system; (b) identifying one or more target genes to include in the gene drive system; (c) recoding the one or more identified target genes in a wild-type background, wherein the recoding further comprises including a new 3'UTR of each target gene in a gene drive cassette; (d) encoding just downstream of the new 3'UTR of the identified gene, one or more guide RNA sequences that correspond to target sites within the wild-type version of the identified gene, wherein the guide RNA sequences are positioned in the gene drive cassette such that the identified gene can drive itself in the presence of an appropriate RNA-guided DNA nuclease; and one or of: (e) including in the gene drive system a second sequence that is sex-determining: (i.) it ensures the organism that includes the gene drive system develops as predetermined sex, or (ii.) it encodes one or more guide RNAs sequences that when expressed disrupt one or more gene(s) and ensures the organism that includes the gene drive system develops as the predetermined sex, or (iii.) it encodes an orthogonal RNA-guided DNA nuclease such that it is expressed exclusively during late meiosis, and also expresses one or more guide RNAs for the orthogonal RNA-guided DNA nuclease that target identified sequences causing chromosomal shredding and ensures that most gametes result in progeny of the predetermined sex. In certain embodiments, a means of identifying the one or more target genes in step (b) comprises a method of any embodiment of any aforementioned aspect of the invention. In some embodiments, a means of recoding the one or more identified target genes in a wild-type background in step (c) comprises a method of any embodiment of any aspect of the invention set forth herein. In some embodiments, a means for (f) identifying one more target sites for chromosomal shredding in step (f) comprises a method of any one of claims using a method of any embodiment of any aspect of the invention set forth herein. In certain embodiments, the gene drive system is based on an RNA-guided DNA-binding protein nuclease.

According to another aspect of the invention, methods of two stage suppression in a gene drive system are provided, the methods including: (a) selecting a gene drive system; (b) identifying one or more target recessive genes corresponding to: sex-specific infertility, infertility, sex-specific viability, or viability of an organism in which the gene drive system will be included; (c) constructing a gene drive system by: (i.) recoding one or more of the identified target genes, wherein the recoded sequence(s) comprise multiple suitable target sites for a subsequent gene drive system with few or no off-targets in the genome and the recoding comprises including a new 3'UTR of each identified target gene in a gene drive cassette; (ii.) encoding just downstream of the new 3'UTR of the identified target gene, one or more guide RNA sequences that correspond to target sites within the wild-type version of the identified target gene, wherein the guide RNA sequences are positioned in the gene drive cassette such that the identified target gene can drive itself in the presence of an appropriate RNA-guided DNA nuclease; (d) preparing one or more organism strains each comprising one of the constructed suppression gene drive systems of step (c); (e) crossing a prepared organism strain of (d) with an N-element daisy chain gene drive strain of the organism and homozygosing offspring of the crossing, wherein offspring of the crossing are complete (N+effector) daisy chain gene drive strain organisms; and (f) preparing a suppressor daisy chain gene drive strain of the organism in which in a wild-type background, one or more of the identified target genes is replaced with an encoded RNA-guided DNA nuclease, and one or more guide RNAs targeting sites are included within the first recoded version of the gene. In some embodiments, a means for constructing a gene drive system in step (i.) comprises a method of any embodiment of any aspect of the invention set forth herein. In some embodiments, a means for preparing the encoded RNA-guided DNA nuclease step (f) comprises a method of any embodiment of any aforementioned aspect of the invention. In certain embodiments, a means for encoding the first recoded version of the gene in step (f) comprises a method of any embodiment of any aspect of the invention set forth herein.

According to another aspect of the invention, methods of two stage suppression in a gene drive system are provided, the methods including (a) selecting a gene drive system; (b) identifying one or more target recessive genes corresponding to: sex-specific infertility, infertility, sex-specific viability, or viability of an organism in which the gene drive system will be included; (c) constructing an N-element gene drive system by: (i.) recoding one or more of the identified target genes, wherein the recoded sequence(s) comprise multiple suitable target sites for a subsequent gene drive system with few or no off-targets in the genome and the recoding comprises including a new 3'UTR of each identified target gene in a gene drive cassette; (ii.) encoding just downstream of the new 3'UTR of the identified target gene, one or more guide RNA sequences that correspond to target sites within the wild-type version of the identified target gene, wherein the guide RNA sequences are positioned in the gene drive cassette such that the identified target gene can drive itself in the presence of an appropriate RNA-guided DNA nuclease; (d) preparing one or more organism strains each comprising one of the constructed suppression gene drive systems of step (c); (e) crossing a prepared organism strain of (d) with an N-element daisy chain gene drive strain of the organism and homozygosing offspring of the crossing, wherein offspring of the crossing are complete (N+effector) daisy chain gene drive strain organisms; and (f) preparing a suppressor daisy chain gene drive strain of the organism in which in a wild-type background, one or more of the identified target genes is replaced with an encoded RNA-guided DNA nuclease, and one or more guide RNAs targeting sites are included within the first recoded version of the gene. In some embodiments, the method also includes (g) sampling a target population of the wild-type organism strain and estimating the number of organisms; (h) releasing a number of the complete (N-element) daisy chain gene drive strain organisms of step (e) at least sufficient to encode a nuclease gene in at least a portion of the target population; (i) sampling strains of organisms collected from the target population following the release and confirming that a suitable fraction of the target population has been recoded; (j) releasing organisms of suppressor daisy chain gene drive strain of prepared in step (f) into the target population, wherein the daisy chain gene drive will spread through and suppress the organisms of the population that were recoded by the release in step (h), but not the wild-type organisms. In some embodiments, the gene drive system is based on an RNA-guided DNA- binding protein nuclease.

According to another aspect of the invention, methods of two-stage suppression in a gene drive system comprising sex -biasing or sex chromosomal shredding, comprising: (a) selecting a gene drive system: (b) identifying one or more first target genes; (c) constructing a gene drive system by: (i.) recoding in a wild-type background, the identified first gene, wherein the recoded sequence contains multiple suitable target sites for a subsequent gene drive system with few or no off-targets in the genome and the recoding comprises including a new 3'UTR of each identified first target gene in a gene drive cassette; and (ii.) encoding just downstream of the new 3'UTR of the identified first target gene, one or more guide RNA sequences that correspond to target sites within the wild-type version of the identified first target gene, wherein the guide RNA sequences are positioned in the gene drive cassette such that the identified first target gene can drive itself in the presence of an appropriate RNA- guided DNA nuclease; (d) preparing one or more organism strains each comprising one of the constructed gene drive systems of step (c); (e) crossing a prepared organism strain of (d) with an N-element daisy chain gene drive strain of the organism and homozygosing offspring of the crossing, wherein offspring of the crossing are complete (N+effector) daisy chain gene drive strain organisms; (f) identifying one or more second target genes; (g) constructing an effector gene drive element by: (i.) recoding in a wild-type background, the identified second gene, wherein the recoded sequence contains multiple suitable target sites within a wild-type strain version of the second identified target gene, a subsequent gene drive system with few or no off-targets in the genome and the recoding comprises including a new 3'UTR of each identified second target gene in a gene drive cassette; and (ii.) encoding just downstream of the new 3'UTR of the identified second target gene, one or more guide RNA sequences that correspond to target sites within the wild-type version of the identified second target gene wherein the guide RNA sequences are positioned in the gene drive cassette such that the identified second target gene can drive itself in the presence of an appropriate RNA-guided DNA nuclease; also encoding one of: a gene that ensures an organism in which it is expressed is of a particular sex or one or more guide RNAs that when expressed result in the organism being of the particular sex; and also encoding: an orthogonal RNA-guided DNA nuclease such that it is expressed exclusively during late meiosis, and two more additional guide RNAs for the orthogonal RNA-guided DNA nuclease that target the sequences causing chromosomal shredding; and optionally (h) preparing one or more organism strains each comprising one of the constructed second gene drive systems of step (g). In some

embodiments, the effector element does not encode guide RNAs for the orthogonal nuclease that direct chromosomal shredding, and instead a second strain encoding a second effector element encoding said guide RNAs is constructed and subsequently released to suppress the population encoding the orthogonal nuclease. In certain embodiments, a means for identifying one or more first target genes in step (b) comprises a method of any embodiment of any aforementioned aspect of the invention. In some embodiments, a means for recoding in a wild-type background, the identified first gene in step (c)(i) comprises a method of any embodiment of any aspect of the invention set forth herein. In some embodiments, a means for identifying one or more second target genes in step (f) comprises a method of any embodiment of any aforementioned aspect of the invention. In certain embodiments, a means of recoding in a wild-type background, the identified second gene in step (g)(i.) comprises a method of any embodiment of any aspect of the invention set forth herein. In some embodiments, the method also includes: (i) sampling a target population of the wild-type organism strain and estimating the number of organisms; (j) releasing a number of the complete (N) daisy chain gene drive strain organisms of step (e) at least sufficient to recode a portion of the genome of at least a portion of the target population; (k) sampling strains of organisms collected from the target population following the release and confirming that a suitable fraction of the target population has been recoded; and optionally (1) releasing organisms of the second daisy chain gene drive strain of step (h) into the target population, wherein the second daisy chain gene drive will spread through and suppress the organisms of the population that were recoded by the release in step (j), but not the wild-type organisms. In some embodiments, wherein the gene drive system is based on one or more RNA-guided DNA-binding protein nucleases.

According to another aspect of the invention, methods preparing a stable population suppression using a daisy chain gene drive, wherein the first element in the daisy drive chain is located in a position unique to one sex thereby suppressing fertility or viability of the other sex are provided, the method including (a) selecting a gene drive system; (b) generating a daisy drive of claims; (c) identifying one or more wild-type sequences present in a currently proximal element of the gene drive system; (d) identifying a genetic element specific to the sex that will not be targeted by the drive system in a strain that includes the daisy drive; (e) encoding one or more guide RNAs that target the wild-type version of the currently proximal element in the daisy drive chain within or adjacent to the sex-specific genetic element to generate a daisy chain gene drive having a first element located in a position unique the sex not targeted by the daisy chain gene drive; and optionally, (f) preparing a cell or organism strain comprising the generated daisy chain gene drive. In some embodiments, a means of generating a daisy drive in step (b) comprises a method of any embodiment of any aspect of the invention set forth herein. In certain embodiments, a means of encoding one or more guide RNAs that target the wild-type version of the currently proximal element in the daisy drive chain within or adjacent to the sex-specific genetic element in step (e) comprises a method of any embodiment of any aspect of the invention set forth herein. In some embodiments, a genetic element specific to the sex that will not be targeted comprises a genetic element specific to males when the drive system disrupts female fertility. In some embodiments, the daisy chain gene drive system is based on one or more RNA-guided DNA- binding protein nucleases.

According to another aspect of the invention, methods of preparing a sex-linked gene drive system that results in sterility of opposite-sex-offspring are provided, the methods including: (a) selecting a gene drive system and a background strain; (b) selecting a sex to be targeted by the gene drive system; (c) determining a number of daisy chain gene drive elements to include in the gene drive system, wherein the number is three or more and is represented as including at least: C-B-A elements; (d) designing and constructing a first organism strain in the background strain, wherein the first organism strain includes only element "A" of the three elements of the daisy chain gene drive and the RNA-guided DNA nuclease is encoded such that the nuclease will be expressed in a zygote and early embryo of the organism strain; (e) identifying a genetic element specific to the sex that is not the sex selected to be targeted by the drive system; (f) encoding in the first organism element "A" strain an element "B" to create a "B-A" daisy drive strain, wherein element "B" comprises one or more guide RNAs selected to target the wild-type version of element "A" that are encoded within or adjacent to the identified sex-specific genetic element, and wherein the element "B" will cause the element "A" to drive; (g) identifying a target gene that is a recessive gene corresponding to sex-specific infertility, (h) creating a second strain by replacing one or more target genes with a sequence encoding one or more guide RNAs targeting the wild-type sequence of the gene, and (i) crossing the prepared second organism strain with the "B-A" daisy drive strain, wherein the offspring of the cross are a sex-specific zygotic daisy drive strain whose opposite-sex offspring are infertile due to loss of both copies of the identified target gene, and wherein the same-sex offspring of the cross will be (B-A- effector) daisy drive organisms of nearly normal fitness. In some embodiments, encoding the element in the initial daisy chain drive such that when expressed, the RNA-guided DNA nuclease will be active and present in a zygote and early embryo of an organism comprising the initial daisy drive comprises inclusion of a constitutive or housekeeping promoter. In certain embodiments, the included promoter is an actin promoter. In some embodiments, the genetic element specific to a sex predetermined to not be targeted by the drive system comprises an element specific to males if the if the drive system is designed to disrupt female fertility of an organism comprising the final gene drive. In certain embodiments, a means for (d) designing and constructing a first organism strain in the background strain in step (d) comprises a method of any one of any embodiment of any aspect of the invention set forth herein, except that the first organism strain includes only element "A" of the three of more elements of the daisy chain gene drive and the RNA-guided DNA nuclease is encoded such that the nuclease will be expressed in a zygote and early embryo of the organism strain. In some embodiments, a means for (f) encoding in the first organism element "A" strain an element "B" to create a "B-A" daisy drive strain, wherein element "B" comprises one or more guide RNAs selected to target the wild-type version of element "A" that are encoded within or adjacent to the identified sex-specific genetic element in step (f) comprises a method of any embodiment of any aspect of the invention set forth herein. In some embodiments, the gene drive system is based on one or more RNA-guided DNA-binding protein nucleases. In some embodiments, the A element encodes two orthogonal RNA- guided DNA nucleases, one of which is expressed in the germline after soma-germline differentiation and corresponds to the guide RNAs in the B element, and the other of which is expressed in the zygote and early embryo and corresponds to the guide RNAs of the effector element.

According to another aspect of the invention, methods of preparing one or more daisy drive elements in which guide RNAs are embedded within introns of target genes are provided, the methods including: (a) selecting a gene drive system; (b) identifying tRNAs for insertion into an intron in a drive element of the selected gene drive system; (c) designing one or more daisy drive elements that are positioned within introns of a target gene, wherein a string of alternating tRNAs and guide RNAs is inserted into an intron, and a tRNA is at each end of the string, and wherein the daisy drive elements comprise two or more recoded nuclease target sites positioned in exons on either side of the intron; and optionally, (d) preparing a daisy chain gene drive strain comprising one or more of the designed daisy drive elements of step (c). In certain embodiments, a means for identifying tRNAs for insertion into an intron in a drive element of the selected gene drive system in step (b) comprises a method of any embodiment of any aspect of the invention set forth herein. In some embodiments, at least two nuclease target sites are positioned in the exons on either side of the intron. In some embodiments, one or more of the guide RNAs in an upstream element of the daisy chain target the recoded nuclease target sites. In certain embodiments, the strain is a cell culture strain. In some embodiments, the strain is an organism strain. In some embodiments, the gene drive system comprises a CRISPR gene drive system. In certain embodiments, the string of alternating tRNAs and guide RNAs is replaced by a string of guide RNAs that do not require external processing factors, such as the crRNAs of Cpf 1. In some embodiments, the tRNAs in the string of alternating tRNAs and guide RNAs are replaced by guide RNAs that do not require external processing factors, such as the crRNAs of Cpfl, and both nucleases are expressed from the appropriate daisy element.

According to another aspect of the invention methods of preparing an evolutionarily unstable yet robust gene drive system through redundancy are provided, the methods including: (a) selecting a gene drive system; (b) identifying two or more target sites within a given gene locus; (c) designing a gene drive system, wherein the gene drive system encodes a single highly active promoter that drives a guide RNA targeting one of the identified target sites and encodes an RNA-guided DNA nuclease that is expressed under conditions for comparatively efficient homologous recombination; and (d) repeating step (c) one, two, or more times, wherein each designed gene drive system targets a different sequence or sequences within the given gene locus and the number of gene drive systems is sufficient to target each of the identified target sites within the given locus, and each drive system is constructed so as to replace the target sites in the given gene locus. In some embodiments, the single highly active promoter in step (b) is identified using a method of any embodiment of any aspect of the invention set forth herein. In some embodiments, a means for encoding the single highly active promoter that drives a guide RNA targeting one of the identified target sites and the RNA-guided DNA nuclease that is expressed under conditions for comparatively efficient homologous recombination, in step (c) comprise a method of any embodiment of any aforementioned aspect of the invention. In certain embodiments, the method also includes: (e) preparing one or more strains of an organism wherein each strain comprises at least one of the gene drive systems designed in step (d). In some embodiments, the method also includes: releasing the prepared organism strains into a local environment. In some embodiments, the inserted DNA replaces all target sites identified within the locus. In certain embodiments, the gene drive strain is a cell line strain. In some embodiments, two or more of the prepared drive systems coexist in a cell. In some embodiments, the gene drive system is based on one or more RNA-guided DNA-binding protein nucleases. According to another aspect of the invention, methods of preparing an evolutionarily unstable yet robust gene drive system through redundancy are provided, the methods including (a) selecting a gene drive system; (b) identifying two or more target sites within a given gene locus; (c) designing a gene drive system, wherein the gene drive system encodes a single highly active promoter that drives a guide RNA targeting one of the identified target sites and encodes an RNA-guided DNA nuclease that is expressed under conditions for comparatively efficient homologous recombination, (d) repeating step (c) one, two, or more times, wherein each designed gene drive system targets a different sequence or sequences within the given gene locus and the number of gene drive systems is sufficient to target each of the identified target sites within the given locus, and each drive system is constructed so as to replace the target sites in the given gene locus; and (e) preparing two or more gene drive strains each comprising one designed gene drive system of step (d); wherein each strain includes one guide RNA per gene drive element that targets a different site within the wild- type locus than is targeted by any of the other prepared gene drive strains. In some embodiments, the single highly active promoter of step (c) is identified using a method of any embodiment of any aspect of the invention set forth herein. In certain embodiments, a means for encoding the single highly active promoter that drives a guide RNA targeting one of the identified target sites and the RNA-guided DNA nuclease that is expressed under conditions for comparatively efficient homologous recombination, in step (c) comprise a method of any embodiment of any aforementioned aspect of the invention. In some embodiments, the prepared gene drive strain is an organism strain. In some embodiments, the method also includes releasing organisms comprising each of the prepared drive systems together. In certain embodiments, the inserted DNA replaces all target sites identified within the locus. In some embodiments, the prepared gene drive strain is a cell line strain. In some embodiments, two or more of the prepared drive systems coexist in a cell of the gene drive cell line or a cell in the organism. In some embodiments, the gene drive system is based on one or more RNA- guided DNA-binding protein nucleases.

According to another aspect of the invention, methods of preparing a double-stranded (ds) DNA sequence capable of producing multiple guide RNAs capable of directing a CRISPR-type protein (complex) to multiple target sites within a cell are provided, the methods including: (a) identifying a divergent plurality of guide RNAs; (b) measuring the activity of the identified guide RNAs; (c) determining 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more highly active guide RNA sequences in the identified plurality of guide RNA sequences; and (d) inserting into a cell, a sequence comprising: a promoter, two or more tRNAs, and two or more of the highly active guide RNAs determined in step (c): two or more tRNA sequences, two more of the determined highly active guide RNA sequences, wherein the highly active guide RNA sequences are expressed from the same single promoter. In certain embodiments, a means for identifying the plurality of divergent guide RNAs comprises a method of any embodiment of any aforementioned aspect of the invention. In some embodiments, a means of measuring the activity in step (b) comprises a transcriptional activity reporter assay. In certain embodiments, the transcriptional activation assay comprises use of a fluorescent reporter and fluorescence-assisted cell sorting to identify a level of guide RNA transcriptional activation. In some embodiments, the promoter is a U6 promoter or functional variant. In some embodiments, the sequence expressed in step (d) comprises: a U6 promoter-tRNAl- sgRNAl-tRNA2-sgRNA2-tRNA3-sgRNA3-tRNA4-sgRNA4, wherein sgRNAl, sgRNA2, sgRNA3, and sqRNA4 are highly active guide RNAs determined in step (c). In certain embodiments, a means for selecting the tRNAs included in the sequence expressed in step (d) comprises a method of any embodiment of any aspect of the invention as set forth herein. In some embodiments, step (d) comprises inserting into a cell a sequence comprising: a promoter and two or more of the highly active guide RNAs determined in step (c), wherein the guide RNAs do not require external processing factors, such as the crRNAs of Cpf 1. In some embodiments, step (c) comprises identifying highly active guide RNAs that do not require external processing and highly active guide RNAs that do require external processing, and part (d) comprises inserting into a cell a sequence comprising: a promoter, two or more guide RNAs of each type arranged so that they alternate, wherein processing of those guide RNAs that do not require external factors liberates pairs comprising one guide RNA of each kind such that both are active.

According to another aspect of the invention, methods of constructing a gene drive system are provided, the methods including one or more methods that include one of more embodiments of any of the aforementioned aspects of the invention.

According to another aspect of the invention, gene drive strains are provided, wherein the gene drive strain is constructed using a means that includes one or more methods that include one of more embodiments of any of the aforementioned aspects of the invention.

Brief Description of One of the Sequences

SEQ ID NO: 1 is an amino acid sequence of an S. pyogenes Cas9 protein sequence [Deltcheva et al., Nature 471, 602-607 (2011)]:

MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET AE ATRLKRT ARRRYTRRKNRIC YLQEIF SNEMAK VDD SFFHRLEE SFL VEEDKKHERH PIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLN PDNSDVDKLFIQLVQTYNQLFEEOTINASGVDAKAILSARLSKSRRLEMJAQLPGEKK NGLFGNLIALSLGLTP FKS FDLAEDAKLQLSKDTYDDDLD LLAQIGDQYADLFL AAK LSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKL REDLLRKQRTFDNG SIPHQIHLGELHAILRRQEDFYPFLKD REKIEKILTFRIPYYVGPLARGNSRFAWMTR KSEETITPW FEEVVDKGASAQSFIERMTNFDK LP EKVLPKHSLLYEYFTVYNELT K VK Y VTEGMRKP AFL S GEQKK AIVDLLFKTNRK VT VKQLKED YFKKIECFD S VEIS G VEDRFNASLGTYFIDLLKIIKDKDFLD EE EDILEDIVLTLTLFEDREMIEERLKTYAH LFDDKVMKQLKRRRYTGWGRLSRKLF GIRDKQSGKTILDFLKSDGF A R FMQLIH DDSLTFKEDIQKAQVSGQGDSLHEfflA LAGSPAIKKGILQTVKVVDELVKVMGRFiK PENrVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEFlPVENTQLQ EKLYL YYLQNGRDMYVDQELDF RLSDYDVDHrVPQSFLKDDSID KVLTRSDK RGKSDN VPSEEVVKKMKNYWRQLLNAKLITQRKFD LTKAERGGLSELDKAGFIKRQLVETR QITKHVAQILDSRMNTKYDElWKLIREVKVITLKSKLVSDFRKDFQFYKVREr NYFiH AFIDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFY SNF NFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNrVKK TEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKS KKLKSVKELLGITF ERSSFEK PIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRM LASAGELQKG ELALPSKYV FLYLASHYEKLKGSPED EQKQLFVEQFIKHYLDEII EQISEFSKRVILADA LDKVLSAYNKHRDKPIREQAENIIHLFTLT LGAPAAFKYFDT TIDRKRYT S TKEVLD ATLIHQ SITGLYETRIDLS QLGGD .

Brief Description of the Drawings

Figure 1 is a schematic diagram illustrating how CRISPR gene drives distort inheritance in a self-sustaining manner by converting heterozygotes into homozygotes in the germline.

Figure 2A-B provides two schematic diagrams illustrating an embodiment of a daisy drive system. Fig. 2A illustrates that a daisy drive system consists of linear daisy chains of serially dependent drive elements. Fig. 2B illustrates that elements at the base of the daisy chain cannot drive and are successively lost over generations, limiting overall spread.

Figure 3 is a schematic diagram showing the family tree of a C- B- A embodiment of a daisy drive over four generations if all organisms mate with wild-type. 14/16 F4 descendants will inherit A, versus 1/16 for a non-drive, 8/16 for a B- A split drive, and 16/16 for a global CRISPR gene drive.

Figure 4A-B provides schematic diagrams showing family tree analysis. Fig. 4A shows results of analysis of a B^A split drive and Fig. 4B is a graphical depiction of total alleles per generation for B- A through D- C- B- A daisy drives. Figure 5 is a schematic diagram illustrating that recombination events that move a guide RNA from one element to another could create a "daisy necklace" capable of self-sustaining global drive. Figure 6 provides a list of sequence-divergent guide RNAs that were designed, constructed, and assayed using the transcriptional activation reporter. The sequences shown are, from top to bottom, SEQ ID NOs: 3-34.

Figure 7 is a schematic diagram illustrating a drive system ensuring that non-homologous end joining ( FEJ) events that repair drive-induced double-strand breaks impair ability to progress through gametogenesis due to disruption of a haploinsufficient gene. In organisms where gametogenesis is proliferative, the cell deficit compensated for by other cells that are not so impaired due to having correctly copied the recoded version accompanying the drive. This can select against potential drive-resistant alleles while reducing or eliminating the fitness cost of incorrect repair. Because total gamete production should be nearly or completely equivalent to wild-type, the organism will not suffer from any fitness cost due to incorrect copying even if the HEJ rate is very low. Guide spacer sequence shown in the top right section of the diagram is SEQ ID NO: 46. Figure 8 is a schematic diagram showing a daisy drive system consisting of a number of serially-dependent elements in which each element in the daisy chain causes the next element to drive. The daisy chain can be of any desired length so long as the total fitness cost is not prohibitive. Figure 9 is a schematic diagram that illustrates redundant targeting of a daisy drive system in which all daisy elements except for the 'A' element encoding the nuclease are inserted at neutral loci that permit expression but do not disrupt important sequences. Because nonhomologous end-joining will create drive-resistant alleles blocking cutting and copying of these daisy elements, each must target both the next element in the chain as well as the nuclease-carrying element in order to ensure that the nuclease element always exhibits drive as long as any daisy element is present. Drive-resistant alleles are consequently not a major problem save for a reduction in drive efficiency, especially with high non-homologous end- joining rates. To compensate for this, additional versions of the drive system (e.g. C2 and B2) can be created with guide RNAs that target different, but nearby sequences within each wild- type neutral locus. As a consequence, the different versions of the drive system will be able to overwrite resistant alleles created by the other versions, reducing the ability of these resistant alleles to impair the spread of the nuclease-carrying element. Both diagrams depict a drive system that also carries an effector element with its own guide RNAs that allow it to drive in the presence of the nuclease gene.

Figure lOA-C provides a schematic diagram and graphs showing embodiments of daisy drive systems employing neutral site targeting as described in Figure 9. Fig. 10A shows an example population on an island to be altered. Fig. 10B illustrates the predicted increase in frequency of the A element depending on the total number of daisy elements from 2-7. Fig. IOC depicts the number of generations required for the A element to spread to near fixation in the target population as a function of release frequency. Each dotted trace corresponds to a different number of daisy elements. The key for number of elements is shown in Fig. 10A. Figures correspond to different rates of homology-directed repair.

Figure 11 A-C provides graphs indicating that the dynamics of a C- B- A embodiment of a daisy drive alleles depends on the seeding frequency and fitness costs. The system does not employ neutral site targeting; rather, each daisy element is constructed such that nonhomologous end-joining is lethal, producing a fitness cost but avoiding all drive-resistant alleles. Fig. 11 A shows that a daisy drive with 2% fitness cost per upstream element and 10% fitness cost for the final element, seeded at 1%, never approaches fixation. Fig. 1 IB shows that the same drive seeded at 5% would rapidly fix in a non-deterministic model. Fig. 11C shows that if the upstream elements cost 10% each, more organisms would need to be released.

Figure 12A-B provides graphs of modelling data illustrating that the A element attains higher frequencies as daisy-chain length increases across a range of fitness costs per upstream element, assuming the final element has a fitness cost of 10%. Again, daisy drive elements are all constructed such that non-homologous end-joining is lethal, in contrast to the neutral sit emodel. Fig. 12A shows that when a population was seeded at a level of 5%, three element chains were sufficient for the A element to reach 99% frequency if the upstream elements have a low fitness cost (2%, left). As the cost increases to 5% (middle), four elements were required, and 10% cost precluded spread above roughly 80%. Fig. 12B illustrates that daisy drives with more elements require fewer organisms to be released in order for the A element to reach a frequency of 99%. Each homing event is assumed to occur with 95% efficiency.

Figure 13A-B provides graphs illustrating that releasing new organisms in each generation enables faster spread and requires fewer organisms per release. Fig. 13 A shows results indicting that three- four- or five-element daisy drives can spread constructs with upstream elements having fitness costs of 2% (left) or 5% (middle) to 99% frequency. Four- or five- element drives are sufficient when the upstream elements have higher (10%) fitness costs. Fig. 13B shows results indicating that repeated release at very low frequency (0.1%) is sufficient for spread of the final element to 99% frequency for upstream elements having fitness costs of 2% (left) or 5% (middle), while >1% repeated release is required for higher cost (10%)) elements.

Figure 14A-B provides a sequence and a graph of results identifying highly active sequence- divergent guide RNAs for SPCas9. Fig. 14A shows a 'Wild-type' sgRNA sequence (SEQ ID NO: 2) that was the template sequence used to generate candidate gRNAs. Fig. 14B shows results of activity assays illustrating relative activities of guide RNAs based on a dCas9-VPR transcriptional activator screen using a tdTomato reporter. Figure 15 is a schematic diagram showing a potential family tree of a C- B- A embodiment of a genetic load daisy drive for which the cargo in the A element disrupts a female fertility gene. The C element is male-linked, ensuring that it does not suffer a fitness cost from the loss of female fertility. Mating events between two parents carrying the A element (boxed) often produce sterile female offspring that will suppress the population.

Figure 16 is a schematic diagram showing a male daisy-drive lineage whose daughters are always sterile, which permits dominant population suppression by titrating the number of males released. Figure 17 is a schematic diagram showing three daisy links used to prepare the C. elegans daisy drive. Daisy link 'A' contained myo3-mCherry-unc54 UTR flanked by 500 bp of both 5' and 3' homology sites for Cku80. Daisy link B contained Pmyo2-GFP-unc54UTR and guides targeting Cku80. It was flanked by both 5' and 3' homology arms to fog2. EM -Hera: Daisy link C contained Prpll28 + BFP + let-858 UTR + gRNA targeting fog-2. Figure 18A-C shows a graphs of raw qPCR reads from Bio-Rad qPCR cfx384. In Fig. 18A the horizontal line in each represents the point at which the Cq, or quantification cycle value was determined.

Figure 19A-C provides three scatter-plot representation of Cq values from the qPCR. The data groups are clearly separated with an average of -1.2 cycles separating the 'Daisy' and 'Control' groups, indicating the drive system was successfully copied due to cutting of the wild-type allele and repair by homologous recombination. Fig. 19A shows results for Daisy Element "A", Fig. 19B shows results for Daisy Element "B", and Fig. 19C shows results for Daisy Element "C".

Detailed Description

Gene drives are genome editing tools that can be used to spread selected genetic modifications through a targeted population of sexually reproducing organisms. Gene drives permit nucleic acid sequences to be introduced into cells, cells lines, and organism strains where they are directed to, and edit, a predetermined gene sequence. Gene drives are named for their ability to "drive" themselves and nearby genes through populations over many generations. Previous RNA-guided gene drive elements based on the CRISPR/Cas9 nuclease could be used to spread many types of genetic alterations through sexually reproducing species (Esvelt, K, et al., 2014 eLife:e03401) These gene drive elements function by

"homing", or the conversion of heterozygotes to homozygotes in the germline, which renders offspring more likely to inherit the gene drive element and the accompanying alteration than Mendelian inheritance would predict. Fig. 1 illustrates how global CRISPR gene drives distort inheritance in a self-sustaining manner by converting heterozygotes into homozygotes in the germline. The self-propagating nature of global gene drive renders the technology uniquely suited to addressing large-scale ecological problems, but tremendously complicates discussions of whether and how to proceed with any given intervention. In addition, there are currently few options for controlling unauthorized or accidentally-released global drive systems.

The invention, in part, relates to novel types of gene drives that are designed to permit controlled, local gene drive activity. The novel control aspects allow release of a gene drive organism strain into a local population with the ability to confine the gene drive organisms such that they only affect local populations and do not risk global gen drive activities. Aspects of the invention, includes methods to design and construct powerful but locally- confined RNA-guided gene drive systems, that are designed to permit local containment of homing drives by arranging CRISPR-based drive components in an interdependent, daisy- chain-like manner, termed "daisy drives".

The invention, in part, includes methods to design, construct and/or use a novel type of gene drive, referred to as a "daisy chain gene drive", which may also be referred to herein as gene drives or daisy drives. The invention, in part relates to methods of designing daisy chain gene drive systems and methods to modify and/or control local populations of organisms by implementing daisy chain gene drive systems of the invention into local populations of organisms. Designing daisy chain drive systems and components thereof, may include one or more methods to select target genes, design, identify, and select active guide RNAs, identify promoter sequences, identify and use spacer sequences, design daisy chain drive elements, select tRNAs or other methods of expressing multiple active guide RNAs, select and use detectable labels, such as fluorescent detectable labels, etc. Certain aspects of the invention include combining one or more of the design and construction methods set forth herein and may also include delivering and implementing a daisy chain gene drive in a cell or organism strain. As used herein the term "daisy chain gene drive" means a gene drive that is includes gene drive components configured in an interdependent, daisy-chain-like manner, termed "daisy drives". In some embodiments of the invention a daisy chain gene drive is a CRISPR-based daisy chain gene drive and includes CRISPR-based drive components in an interdependent daisy chain configuration. Fig. 2 illustrates a general design strategy for daisy chain gene drives of the invention. A daisy drive system of the invention consists of a linear series of genetic elements in which each element drives the next in the daisy chain. Fig. 2A illustrates one embodiment of a daisy chain drive that includes three elements, C- B- A. The final element in the chain (the "cargo") is driven to higher and higher frequencies in the population by the elements below it in the chain, much like the cargo of a rocket is driven by the booster stages below (Fig. 2B). Because the element at the base of the daisy chain never exhibits drive, basal elements are progressively lost over generations. The more elements to a daisy drive, the higher the frequency of the cargo will be lifted. The term "cargo" may be used interchangeably with the term "payload" herein.

A daisy chain drive system designed using one or more methods of the invention can recapitulate any effect accessible to a global CRISPR gene drive, including either population alteration or suppression. A daisy chain drive designed, constructed, and/or implemented using one or more methods of the invention, permits the spread of an effector element through a population to be enhanced by including daisy chain gene drive components. For example, but not intended to be limiting, an effector element could be enhanced by adding a daisy chain gene drive including elements C- B- A, wherein the effector exhibits drive in the presence of A, A exhibits drive in the presence of B, and B exhibits drive in the presence of C. Family tree analysis indicates that with such a gene drive design there will be many more copies of A relative to those generated using a previous gene drives designs, such as B- A split drives. Fig. 3 shows results of family tree analysis that demonstrates the power of including an additional daisy drive element to spread the cargo to more offspring in the F4 generation. Fig. 4A-C illustrates the strength of a daisy chain gene drive of the invention, compared to a split gene drive system.

Aspects of the invention are based, in part, on the design and construction of daisy chain gene drives, and their use in cells, cell lines, and organisms as nuclease-based evolutionarily stable gene drive systems that are capable of altering or suppressing populations of organisms. Certain embodiments of daisy chain gene drives designed and prepared using methods of the invention include RNA-guided DNA binding proteins that when expressed in a cell co-localize with guide RNA at a target DNA site and act as gene drives. Dai sy chain gene drive systems of the invention may be used to edit the genome of a host (target) cell or organism into which components of the daisy chain gene drive are delivered. As used herein, the terms "used" and "implemented" when used in reference to daisy chain gene drives, means a designed and constructed daisy chain gene drive is included in a cell or organism strain. It will be understood that implementation of a daisy chain gene drive may occur in one event or may be a multi-part implementation.

A daisy chain gene drive system that may be designed, constructed, and implement using one or more methods of the invention, is an RNA-guided DNA-binding protein endonuclease daisy chain gene drive system. Components of gene drive systems (for example: drive elements, guide RNAs, expression cassettes, vectors, endonucleases, promoters, DNA binding proteins, etc.) and methods for preparing and using such

components, are known in the art and may be used in conjunction with methods of the invention to design, construct, and implement daisy chain gene drives of the invention, see for example: DiCarlo, J.E. et al., Nat Biotechnol. 2015 Dec;33(12): 1250-1255; Gantz V.M. & E. Bier Science, 2015 Apr 24;348(6233):442-4.; Gantz V.M. et al., Proc Natl Acad Sci U S A. 2015 Dec 8; 112(49); and Hammond, A. et al., Nat Biotechnol. 2015 Dec 7;

doi: 10.1038/nbt.3439; the content of each of which is incorporated by reference herein in its entirety. In addition, methods and components of split-drive gene drives are known in the art and may be used in conjunction with methods described herein to design, construct, and implement daisy chain gene drives of the invention, see for example: Esvelt K. et al., eLife 2014;3 :e03401; DiCarlo, J.E. et al., Nat Biotechnol. 2015 Dec;33(12): 1250-1255; and Akbari et al., Science, 30 July 2015 (10.1126/science.aac7932) the content of each of which is incorporated by reference herein in its entirety.

Embodiments of certain RNA-guided DNA-binding protein endonuclease daisy chain gene drive systems of the invention include aspects of CRISPR systems. Details of CRISPR systems such as CRISPR-Cas systems and examples of their use are known in the art, see for example: Deltcheva, E. et al. Nature 471, 602-607 (2011); Gasiunas, G., et al., PNAS USA 109, E2579-2586 (2012); Jinek, M. et al. Science 337, 816-821 (2012); Sapranauskas, R. et al. Nucleic acids research 39, 9275-9282 (2011); Bhaya, D., et al., Annual review of genetics 45, 273-297 (2011); and H. Deveau et al., Journal of Bacteriology 190, 1390 (Feb, 2008), the content of each of which is incorporated by reference herein in its entirety.

Three classes of CRISPR systems are generally known and are referred to as Type I, Type II or Type III. According to one aspect of the invention, methods to design and/or construct a daisy chain gene drive may include features of one or more of the three classes of CRISPR systems. Type I, II, and III CRISPR systems and their components are well known in the art. See for example, K. S. Makarova et al., Nature Reviews

Microbiology 9, 467 (Jun, 2011); P. Horvath & R. Barrangou, Science 327, 167 (Jan 8, 2010); H. Deveau et al., Journal of Bacteriology 190, 1390 (Feb, 2008); J. R. van der Ploeg, Microbiology 155, 1966 (Jun, 2009), the contents of each of which is incorporated by reference herein in its entirety. Bioinformatic analyses have generated extensive databases of CRISPR loci in a variety of bacteria that maybe used in conjunction with methods of the invention to design and construct daisy chain gene drives. See for example: M. Rho, et al., PLoS genetics 8, el002441 (2012) and D. T. Pride et al., Genome Research 21, 126 (Jan,

2011) each of which is incorporated by reference herein in its entirety. A recently designated Type V system is similar in many aspects to Type II systems and may be relevant for genome editing and therefore gene drive systems (B. Zetsche et al., 2015, Cell 163, 1-13; T. Yamano et al., 2016, Cell, April 21 doi: 10.1016/j .cell.2016.04.003; D. Dong et al., 2016, Nature, 20 April, doi: 10.1038/nature 17944; I. Fonfara et al., 2016, Nature, 20 April,

doi: 10.1038/naturel7945). It will be understood that references herein to "Cas9", the RNA- guided DNA-binding protein nuclease of Type II CRISPR systems, can be replaced by "Cpfl", the RNA-guided DNA-binding protein nuclease of Type V systems. It will be understood, as described elsewhere herein, certain embodiments of daisy chain gene drives of the invention may include a targeted DNA-binding nuclease other than an RNA-guided DNA-binding nuclease. For example, in some embodiments a daisy chain gene drive may include a nucleic acid-guided DNA binding nuclease such as a DNA-guided DNA-binding nuclease (see Gao, F., et al., Nature Biotech online publication, May 2, 2016:

doi: 10.1038/nbt.3547, the content of which is incorporated herein by reference).

Drive Systems General Strategies for Design, Construction, and Deployment

A daisy chain drive system includes a linear series or "chain" of genetic elements in which each element drives the next element in the daisy chain (Fig. 2A). A daisy chain drive system designed using methods of the invention can be introduced into a population of organisms and the "cargo" or top of the chain is driven to higher and higher frequencies in the population by the elements below it in the chain. Because the element at the base of the daisy chain never exhibits drive, the base elements in the chain may be progressively lost over generations. The more elements to a daisy drive, the higher the frequency of the cargo in the population. A non-limiting example of a daisy chain drive system is a drive that includes three genetic elements, and is represented as C- B- A. In the example daisy chain drive, the RNA-guided DNA-binding protein nuclease gene used by the drive system to bias inheritance is encoded in the "A" element, while the element at the base of the chain is element "C". It will be understood that additional elements, represented as elements D, E, F, G, etc. may be included in a daisy chain drive designed and constructions using methods of the invention, non-limiting examples of which are daisy drives D- C-^B- A, E- D^C^B^A, and F^ E^ D^C^B^A, etc. As used herein, the letters A, B, C, D, E, F, etc. each represents a different element in a daisy chain designed and/or constructed using methods such as those disclosed herein.

It has now been identified that the spread of an element A in a gene drive may be enhanced by adding additional links in the daisy chain of gene drive components, e.g.

C- B- A. Thus, inclusion of a daisy chain gene drive such as C- B- A in a population of an organism will result in many more copies of "A" relative to the number that would result from a inclusion of a B- A split drive. Assuming fitness neutrality and ignoring stochastic effects, a comparatively small release of organisms encoding an C- B- A daisy drive will result in a fixed incidence of C, increasing B, and a more rapidly increasing A. The "chain" of a daisy chain drive can be extended indefinitely, e.g. D- C- B- A etc. to achieve successively more powerful local drive. Thus in some aspects of the invention a daisy chain gene drive includes at least 3, 4, 5, 6, 7, 8, 9 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more elements. A daisy chain gene drive system may be designed using methods of the invention to include a linear series of genetic elements in which each element causes the one, immediately downstream to exhibit drive, for example, though not intended to be limiting: a daisy gene drive with elements D- C- B- A, of which the furthest upstream element is D and the furthest downstream element is A. Sequential letters are used herein to designate daisy chin gene drive "elements", but it will be understood that alternative symbols can be used if desired, for example numbers, or other desired nomenclature.

Certain embodiments of the invention include methods that can be used

independently, or in combination with each other or with other gene drive design methods, to prepare daisy chain gene drives that can be used to construct powerful and locally-confined RNA-guided drive systems. Daisy chain gene drives designed using one or more methods of the invention, can be delivered into cells, cell lines, and/or organisms where they act to edit the genome in a stable, controlled manner. Daisy chain drive systems designed using one or more methods of the invention may be utilized in stable genome-modifying applications for which global drive systems and/or existing local drives are unsuitable. For example, methods of the invention can be used to prepare one or more "daisy chain drive" organism strains that may then be released into a local wild population of the organism. The presence of the daisy chain drive organisms as a predetermined small fraction of a local wild population of the organism can be used to drive a useful genetic element, included in the drive, to local fixation for a wide range of fitness parameters without resulting in global spread. Daisy chain gene drives designed using methods of the invention may permit local communities to decide whether, when, and how to alter shared regional ecosystems.

Methods of the invention, in some aspects, include design, construction, and use of daisy chain gene drives systems that include a "generic" daisy chain gene drive. As used herein, a generic daisy chain gene drive includes N elements. In embodiments of an N- element daisy chain gene drive, the terminal element in the chain (designated the A element) encodes an RNA-guided DNA nuclease. Additional effector elements that modulate the population are not included in the generic N-element daisy chain gene drive. An N+effector- element daisy chain gene drive of the invention can be utilized in a number of different methods for modulating gene expression and organism populations. One non-limiting example is delivery into an organism that includes a "generic" N-element daisy chain gene drive, an "effector" element designed to accomplish a desired genome modulation (for example gene alteration or suppression) that also encodes guide RNAs that enable "effector" to drive in the presence of the RNA-guided DNA nuclease (encoded in the "A" element). In this scenario, the "effector" element may be added to the organism's genome directly by standard methods known to those in the art so as to create a complete N+effector-element daisy chain drive organism, that is effective to accomplish the desired genome modulation. In another non-limiting example, a "generic" N-element organism strain is prepared and another organism of the same species background is prepared that includes an "effector" element designed to accomplish a desired genome modulation, such as gene alteration or suppression, and that also encodes guide RNAs that enable "effector" to exhibit drive in the presence of the RNA-guided DNA nuclease (encoded in the "A" element). The organism strain comprising the N-element daisy chain gene drive is crossed with the "effector" element containing organism strain thereby creating offspring that are complete N+effector-element daisy drive organisms. In another non-limiting example, organisms that include the designed "generic" N-element daisy chain gene drive may be released into an environment to initiate a daisy chain drive effect that spreads the gene encoding the RNA-guided DNA nuclease (encoded in element "A") through a local population of the wild-type organism, after which one or more organisms of the same background strain as the N-element organisms, but that include an element encoding another desired genome modulation effect, for example alteration or suppression, (designated as a "effector" element) can be released into the N- element daisy chain gene drive organism population to accomplish the desired genome modulation effect. It will be understood that if the desired gene modulation effect is suppression, the release will largely eliminate the RNA-guided DNA nuclease from the population, and if the desired genome modulation is alteration or gene expression, it can be accomplished two or more times in series or in parallel by releasing into the N-element organism population, two, three, four, five, six, seven, or more organism strains prepared such that each includes a different effector element.

C ertain aspects of the invention include methods of preparing cells, cell lines, and/or organisms that include daisy chain gene drives that encode Cas9 or other CRISPR nuclease proteins such as Cpf 1. In some embodiments of such a daisy chain gene drive system, methods of the invention can be used to design, construct and use one 'generic' daisy chain drive strain per organism species. Using such a strategy, one or more "effector" elements carrying genetic cargo can be added directly to the generic daisy chain drive strain, wherein each "effector" element also encodes guide RNAs sufficient to drive itself in the presence of the expressed Cas9 or other CRISPR nuclease. This non-limiting example of single-strain, single-stage approach can be designed, constructed, and implemented using methods of the invention. Another method of the invention may include preparing a generic daisy drive organism strain that includes the CRISPR nuclease gene, and is released into a target region resulting in the spread of the CRISPR nuclease gene through a population of the organism in the target region. One or more additional organism strains can be prepared in the same wild- type organism strain as the generic daisy drive organism strain, but that don't include the N- element generic daisy chain gene drive, but that do include one or more different "effector" elements each designed to produce an desired effect on a selected target gene. The "effector" element strain can also be released into the target region and matings between "N-element" strain organisms and "effector" element strain organisms result in offspring that include both the "N" and "effector" elements, and the presence of the full "N+effector" daisy chain gene drive produces the desired effect on the preselected target gene(s). This non-limiting example of a multi-strain, single-stage approach can be designed, constructed, and implemented using methods of the invention.

Another embodiment of the invention includes preparing a generic (N-element) daisy chain drive strain that is released into a region in the wild and the spread of the CRISPR nuclease gene in the region can be monitored. The monitoring results identify the exact region that was affected by the release. Optionally, spread within this region may be adjusted by releasing wild-type organisms, thereby shifting the ratio of the N-element organism strain to the wild-type organism strain. When acceptable release numbers and parameters have been determined, a subsequent release of daisy chain drive strains carrying "effector" elements that have been designed to produce a desired effect on a selected target gene, would then initiate the desired effect. Methods of the invention to design, construct and implement daisy chain gene drives and systems, may be used in additional strategies for population control.

Gene Drive Components

Aspects of the invention include methods of preparing cells, cell lines, and/or organisms that include daisy chain gene drives. Daisy chain gene drives that may be delivered into a cell or organism may be designed and constructed using embodiments of methods of the invention. Design methods of the invention are directed to genome editing systems comprising components that can be separately encoded as nucleic acid sequences that are delivered into the genome a cell or organism. A daisy chain gene drive system that may be designed using methods set forth herein may include one or more of the design, construction, and testing of one or more components of the daisy chain gene drive, including, but not limited to: guide RNAs, guided DNA binding proteins, nucleic acid-guided DNA binding proteins, RNA-guided DNA binding proteins, DNA-guided DNA binding proteins, promoter/enhancer/3'UTR sequences, housekeeping gene sequences, promoter sequences, predetermined target genes, tRNA sequences, and sequences encoding detectable labels, such as but not limited to fluorescent labels.

Design methods of the invention may be applied when a gene drive system has been selected and in some embodiments include identification of a target gene in the genome of a host cell or organism into which the gene drive will be delivered. As used herein the term "host" or "target" when used in reference to a cell, cell line or organism, means a cell, cell line, or organism, respectively that includes a daisy chain gene drive designed using one or more methods of the invention. In some embodiments of the invention, a host cell is a germline cell.

Target Genes

Target genes, also referred to herein as target nucleic acids, may include any nucleic acid sequence having an effect that is of interest to be modulated using a daisy chain gene drive of the invention. In some aspects of the invention a target gene comprises DNA, which may be double-stranded DNA or single-stranded DNA. A gene selected as target gene in a daisy chain gene drive may be a nucleic acid sequence in the genome of a host cell. A daisy chain gene drive of the invention may, in some aspects of the invention, be designed such that it includes a gene drive cassette comprising one or more of: a promoter/enhancer/3'UTR sequence, a nucleic acid-guided DNA binding protein, an RNA-guided DNA binding protein gene sequence, and one or more RNA guide sequences. When expressed in a host cell the promoter/enhancer/3'UTR may drive expression of the RNA-guided DNA binding protein gene, which, in conjunction with the RNA guide sequences is directed to the selected target gene. The promoter/enhancer/3'UTR may be inserted along with the encoded RNA-guided DNA binding protein nuclease gene sequence anywhere in the genome, but is typically inserted downstream of the 3'UTR of a target gene. Alternatively, the RNA-guided DNA binding protein nuclease gene sequence may be inserted along with a 2A fusion peptide to a target gene with the desired expression characteristics such that it is transcriptionally and translationally coupled to that gene; in such a case the target gene is typically recoded adjacent to the 2 A fusion. One or more design methods of the invention can be used to identify and select a target gene, and to design guide RNAs having a sufficient level of activity and specificity to guide and position a DNA binding protein to a nucleic acid sequence adjacent, or in close proximity, to the target gene sequence. In certain daisy chain gene drives an expressed DNA binding protein has nuclease activity and when positioned in relation to the target gene, a DNA binding protein cuts the target gene and disrupts the normal effect/action of the target gene in the cell.

Assays described herein, and others known in the art, can be used to determine whether a designed guide RNA and DNA binding protein complex binds to or co-localizes with the host DNA in a manner in that results in a desired effect on the target nucleic acid. For example, though not intended to be limiting, if a desired effect on a target gene is to inhibit or suppress a target gene's expression, assays can be performed to determine whether or not the one more designed guide RNAs and DNA binding proteins, is effective to reduce transcription or expression of the target gene. As a non-limiting example, a transcription activity reporter assay described elsewhere herein may be used to determine whether a designed guide RNA and DNA binding protein have a desired effect on a selected target gene.

In some aspects of the invention a target gene is a haploinsufficient gene, which is a gene for which a single copy is insufficient for normal growth and division of a cell in which it is located. A target gene useful in daisy chain gene drives of the invention may also be a recessive gene, and the action or function of altering or disrupting the gene may correspond to: sex-specific infertility, infertility, sex-specific viability, or viability. In some aspects of the invention a target gene is a gene encoding a ribosomal protein. In certain aspects of the invention, a target gene may include nucleic acid sequences present on either side of an intron. In some methods of the invention, art-known haploinsufficient genes may be used to design, construct, and implement a daisy chain gene drive system of the invention. In certain aspects of the invention, a review of the scientific literature and/or application of routine genetic testing techniques can assist in identifying suitable candidate target genes. Methods are provided herein to identify and test candidate target genes for use in designing, constructing, and implementing daisy chain gene drives of the invention.

It will be understood that selecting a target gene for inclusion in a daisy chain gene drive of the invention may be based, at least in part, on the role of the target gene in the daisy chain gene drive. For example, in certain aspects of daisy chain gene drives of the invention, a target gene may be selected for a "drive element", non-limiting examples of which are: a non-"A" element, an "A" element carrying a nuclease gene, an "A" element that coordinates drive of a number of different changes that result from the daisy chain gene drive system, or an effector element that edits the sequence of or outright disrupts the target gene. For such drive elements, non-limiting examples of suitable genes for selection are haploinsufficient genes and genes that are important for fitness of the host cell or organism. In some aspects of daisy chain gene drives of the invention, a target gene may be selected for a "cargo element", non-limiting examples of which include: an "A" element and a gene that is one of a set of genes altered by simultaneous changes that result from the daisy chain gene drive system. For such cargo elements, non-limiting examples of suitable genes for selection are: any gene, but which may be a gene that is important for fitness of the host cell or organism, a gene to be suppressed, and a gene that is important in fertility and/or viability of the host cell or organism, as described elsewhere herein.

In some aspects of the invention, a target gene is a large ribosomal subunit gene and in certain aspects of the invention, a target gene is a small ribosomal subunit gene. For example, though not intended to be limiting: a target gene may be one of: RpLl, RpL2, RpL3, RpL4, RpL5, RpL6, RpL7, RpL8, RpL9, RpLlO, RpLl l, RpL12, RpL13, RpL14, RpL15, RpL16, RpL17, RpL18, RpL19, and. RpL20. Additional art-known large ribosomal subunit genes and variants thereof are suitable as target genes in methods of the invention. Other non-limiting examples of target genes are: RpSl, RpS2, RpS3, RpS4, RpS5, RpS6, RpS7, RpS8, RpS9, RpSlO, RpSl l, RpS12, RpS13, RpS14, RpS15, RpS16, RpS17, RpS18, RpS19, and. RpS20. Additional art-known small ribosomal subunit genes and variants thereof, large ribosomal subunit genes and variants thereof, and other genes and variants thereof, are suitable as target genes in methods of the invention.

DNA Binding Proteins

Components of a gene drive system designed using at least one method of the invention may include DNA binding proteins and functional variants thereof. In certain aspects of the invention, a DNA-binding protein may be a nucleic acid-guided DNA binding protein. Non-limiting examples of types of nucleic acid DNA-binding proteins that may be used in some embodiments of daisy chain gene drives of the invention include: RNA-guided DNA-binding proteins and DNA-guided DNA-binding proteins. DNA binding proteins are known in the art, and include, but are not limited to: naturally occurring DNA binding proteins, a non-limiting example of which is a Cas9 protein, which has nuclease activity and cuts double stranded DNA. Cas9 proteins and Type II CRISPR systems are well documented in the art. (See for example, Makarova et al., Nature Reviews, Microbiology, Vol. 9, June 2011, pp. 467-477, the content of which is incorporated by reference herein in its entirety.) As used herein, the term "DNA binding protein having nuclease activity" refers to DNA binding proteins having nuclease activity and also functional variants thereof. SEQ ID NO: 1 is an amino acid sequence of Cas9, and may be used in methods of the invention as an RNA- guided DNA binding protein having nuclease activity. Functional variants of SEQ ID NO: 1 can also be used in daisy chain gene drives designed, constructed, and/or implemented using one or more methods of the invention. A functional variant of SEQ ID NO: 1 differs in amino acid sequence from SEQ ID NO: 1, referred to as the variant's "parent" sequence, while retaining from a least a portion to all of the nuclease activity of its parent protein.

In some embodiments, a daisy chain gene drive of the invention may include a DNA- guided DNA-binding nuclease. Information on identification and use of DNA-guided binding proteins, for example in DNA-guided genome editing systems, is available in the art (Gao, F., et al., Nature Biotech online publication, May 2, 2016: doi: 10.1038/nbt.3547, the content of which is incorporated herein by reference in its entirety).

A DNA binding protein having nuclease activity function to cut double stranded DNA that may be used in aspects of methods of the invention can include DNA binding proteins that have one or more polypeptide sequences exhibiting nuclease activity. A DNA binding protein with multiple regions that have nuclease activity may comprise two separate nuclease domains, each of which functions to cut a particular strand of a double-stranded DNA.

Polypeptide sequences that have nuclease activity are known in the art, and non-limiting examples include: a McrA-HNH nuclease related domain and a RuvC-like nuclease domain, or functional variants thereof. In S. pyogenes, a Cas9 DNA binding protein creates a blunt- ended double-stranded break that is mediated by two catalytic domains in the Cas9 binding protein: an HNH domain that cleaves the complementary strand of the DNA and a RuvC-like domain that cleaves the non-complementary strand. [See Jinke et al., Science 337, 816-821 (2012), the content of which is incorporated by reference herein in its entirety]. Cas9 proteins are known to exist in many Type II CRISPR systems, see for example, Makarova et al., Nature Reviews, Microbiology, Vol. 9, June 2011, pp. 467-477, supplemental information, the content of which is incorporated herein by reference in its entirety. The Cas9 protein may be referred by one of skill in the art in the literature as Csnl . Alternatives to Cas9 include but are not limited to Cpf 1 proteins from Type V CRISPR systems (See for example Zetsche et al., Cpfl Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR- Cas System, Cell (2015), //dx.doi.org/10.1016/j .cell.2015.09.038). In certain aspects of the invention, a daisy chain gene drive may include a DNA binding protein that does not have nuclease activity. Guide Nucleic Acids

Methods of the invention, in part, include design, construction, and implementation of daisy chain gene drives that include guide nucleic acid molecules, non-limiting examples of which are guide RNAs and guide DNAs. Information relating to guide DNAs can be found in Gao, F., et al., Nature Biotech online publication, May 2, 2016: doi: 10.1038/nbt.3547, the content of which is incorporated herein by reference in its entirety. Guide RNAs are also referred to herein as short guide RNAs, sgRNAs, and gRNAs, as well as crRNAs for certain nucleases such as Cpfl . A guide RNA is designed and selected such that it is complementary to a DNA sequence of the selected target gene in the genome of a cell, and so the guide RNA acts in complex with a DNA binding protein, or variant thereof to direct degradation of the complementary sequence within the target gene.

In some aspects of the invention methods are provided that can be used to prepare a gene drive in which an exogenous nucleic acid sequence is delivered into a host cell, and is expressed in the cell to produce a nucleic acid-guided DNA binding protein having nuclease activity, and one or more guide nucleic acids. In a non-limiting example: a vector comprising a sequence encoding the one or more guide RNAs and the RNA-guided DNA binding protein may be designed and used in daisy chain gene drives of the invention. Expression of the vector sequences in the host cell results in production of a complex of the RNA-guided DNA binding protein and guide RNAs that is directed by the guide RNA(s) to the preselected target gene, where the complex co-localizes to, or bind with, the target gene and he target gene is cleaved in a site-specific manner by the nuclease activity of the RNA guided DNA binding protein.

Methods of designing guide RNAs to direct an RNA-guided DNA binding protein to a selected target gene are provided herein. Guide RNAs can be designed, prepared, tested, and selected for use in a daisy chain gene drive system of the invention using one or more of the methods provided herein, in conjunction with knowledge in the art relating to DNA binding, vector preparation and use, RNA-guided DNA binding proteins, CRISPR system components and implementation, etc. It has not previously been possible to produce highly divergent guide RNAs and methods are provide herein that permits the design, construction, and implementation of active divergent guide nucleic acids. For example, though not intended to be limiting, methods of the invention may be used to design, construct, and implement a plurality of diverse/divergent guide RNA for an RNA-guided DNA nuclease. Methods of the invention also can be used to determine activity of the divergent guide nucleic acids, for example, though not intended to be limited, using a fluorescent reporter assay such as a procedure set forth herein in Method 1.1. It has previously been very difficult to synthesize repetitive sequences, which has precluded attempts to quickly make DNA sequences capable of expressing multiple guide RNAs. Methods of the invention permit rapid identification and preparation of minimally repetitive sequences encoding guide RNAs. As used herein, the term "elements" when used in the context of preparing divergent guide RNA sequences means the backbone sequence of the guide RNA that is recognized by the nuclease and is capable of directing the nuclease to cut a predetermined target sequence.

Non-limiting examples of guide RNAs designed using methods of the invention are set forth herein as SEQ ID NO: 3-34. The length of a guide RNA designed using methods of the invention may be at least 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, and 500 base pairs, including all integers between those listed. It will be understood that a maximum or minimum permissible length of a guide RNA is limited to a length at which the guide RNA functions as a guide RNA in a daisy chain gene drive of the invention.

Design and Use of Divergent RNA sequences

The invention, in part, also relates to methods of preparing a readily synthesized double-stranded (ds) DNA sequence that can be used to produce multiple guide RNAs. The produced multiple (or plurality of) guide RNAs can prepared such that they are able to direct a CRISPR-type protein (complex) to multiple target sites within a cell. Methods of the invention can be used to prepare divergent guide RNA sequences and the use of divergent guide RNA sequences results in the ability to target a number of targets sites within the same cell. Divergent sequences may be prepared using methods of the invention for use in daisy chain gene drives as disclosed herein, and also for other uses in cells and organisms. For example, methods to design and prepare divergent guide RNA sequences can be used to prepare a plurality of sequences that have minimal sequence homology/identity between themselves and so can be used for multi-targeting. As used herein, the term "multi -targeting" when used in the context of a plurality of divergent sequences means that the sequences are designed such that they target multiple different sequence sites, for example in a cell in which they are expressed.

It has previously been prohibitively difficult to synthesize repetitive sequences.

Methods disclosure herein may be used to obviate this difficulty and permit rapid preparation of DNA sequences capable of expressing multiple guide RNAs. In some aspects of the invention, available information on sequences of interest is used to create a map or diagram of guide RNA that shows each possible individually accepted change throughout the structure of the guide RNA. After acceptable sequence changes have been mapped and identified, several 5, 10, 15, 20, 25, 30, 35, 40, 45, or more elements are designed that combine different combinations of the of these accepted changes. The elements are designed to minimize the length of sequences that are shared between the designed elements. Thus, the elements are designed to minimize the length of any sequences common to two or more of the designed elements. As used herein, the term "element" when used in the context of preparing divergent nucleic acid sequences, such as divergent guide RNA sequences, means the backbone sequence of the guide RNA that is recognized by a preselected nuclease and that is capable of directing the nuclease to cut a preselected target sequence.

The activity and functionality of the designed backbones of the guide RNA sequences is determined and those that have high activity are selected. The activity of the designed divergent sequences can be tested using transcription assays such as those disclosed herein, or using other art-known assays. The activity of the guide RNA is also referred to herein as "function" of the guide RNA. Thus, a guide RNA that has a high activity is one that functions in a desired manner, for example: to be recognized by a nuclease and directing the nuclease to a preselected target gene sequence. Identified high-activity guide RNAs can be used in methods of the invention to construct evolutionarily stable homing-based gene drive systems that target multiple sites to overcome the evolution of mutations that block cutting. Divergent guide RNAs prepared using methods of the invention significantly reduce the chance of recombination between homologous sequences within the drive cassette, which is a major problem for highly repetitive drive cassettes (Simoni et al Nucl. Acids Res. 2014 //dx. doi.org/10.1093/nar/gku387), the resulting drive system will be stable.

An example of the method of preparing divergent sequences, includes, but is not limited to: identifying divergent guide RNAs with high activity using methods described above and also in Method 1.0 and expressing multiple guide RNAs from a single promoter using tRNA processing [see Xie et al. (2015) PNAS doi: 10.1073/pnas.1420294112 , Port and Bullock (2016) bioRxiv doi: 10.1101/046417, the content of each of which is incorporated herein in its entirety]. The guide RNA sequences and tRNA sequences can be synthesized along with a promoter that has been identified to work well in a target organism in which the guide RNAs will be implemented. A non-limiting example of a promoter that may be included is a U6 promoter or equivalent. Non-limiting examples of a sequence of a promoter, tRNAs, and a plurality of divergent guide RNAs are: U6 promoter-tRNAl-sgRNAl-tRNA2- sgRNA2-tRNA3-sgRNA3-tRNA4-sgRNA4; promoter-tRNAl -sgRNAl -tRNA2-sgRNA2- tRNA3 -sgRNA3 -tRNA(N)-sgRNA(N), wherein "N" is the highest number in the series, for example, if there are four tRNAs and four sgRNAs, the series would be: promoter-tRNAl- sgRNAl-tRNA2-sgRNA2-tRNA3-sgRNA3-tRNA4-sgRNA4, if there are six tRNAs and six sgRNAs the series would be: promoter-tRNAl -sgRNAl -tRNA2-sgRNA2-tRNA3-sgRNA3 - tRNA6-sgRNA6. "N" may be independently determined for sgRNAs and tRNAs. "N" may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more. Other non- limiting examples of a sequence expressing multiple guide RNAs with minimal sequence homology include: a U6 promoter-crRNAl-crRNA2-crRNA(N), wherein crRNA 1, crRNA2, crRNA3, and crRNA(N) are highly active guide RNAs determined in step (c), and "N" is the highest number in the series Synthesis of the designed DNA can be done using art-known methods or obtained from commercial users of the art such as, but not limited to: Integrated DNA Technologies gBlocks (Integrated DNA Technologies, Coralville, Iowa) and

ThermoFisher GeneArt Strings (Thermo Fisher Scientific).

Methods for preparing a plurality of divergent nucleic acid sequences as set forth herein in reference to preparing divergent sequences for daisy chain gene drives, can also be used to prepare divergent sequences for use in other multiplexing methods, including but not limited to gene drive methods. The resulting sequences can be used to target multiple target sequences. Additional Components

Methods of the invention, in part, include design, construction, and use of additional sequences that may be included in a vector delivered to a cell as part of a daisy chain gene drive. Sequences such as: promoter sequences, enhancer sequences, 3' untranslated region (3'UTR) sequences can be included. Those skilled in the art will understand how to use such sequences to design, construct, and implement daisy chain gene drives of the invention based on methods, components, and strategies disclosed herein and art-known gene drive methods and components.

Variants

Components of a daisy chain gene drive may include sequences described herein, or designed using one or more methods of the invention and may also include functional variants of such sequences. A variant polypeptide may include deletions, point mutations, truncations, amino acid substitutions and/or additions of amino acids or non-amino acid moieties, as compared to its parent polypeptide. Modifications of a polypeptide of the invention may be made by modification of the nucleic acid sequence that encodes the polypeptide. The terms "protein" and "polypeptide" are used interchangeably herein as are the terms "polynucleotide" and "nucleic acid" sequence. A nucleic acid sequence may comprise genetic material including, but not limited to: RNA, DNA, mRNA, cDNA, etc. As used herein with respect to polypeptides, proteins, or fragments thereof, and polynucleotides that encode such polypeptides the term "exogenous" means the one that has been introduced into a cell, cell line, organism, or organism strain and not naturally present in the wild-type background of the cell or organism strain.

In certain embodiments of the invention, a polypeptide or nucleic acid variant may be a polypeptide or nucleic acid, respectively that is modified from its "parent" polypeptide or nucleic acid sequence. Variant polypeptides and nucleic acids can be tested for one or more activities (e.g., delivery to a target gene, suppression of a target gene, etc.) to determine which variants are possess desired functionality for use in a daisy chain gene drive of the invention.

The skilled artisan will also realize that conservative amino acid substitutions may be made in a polypeptide, for example in a Cas9 polypeptide, to design and construct a functional variant useful in a daisy chain gene drive of the invention. As used herein the term "functional variant" used in relation to polypeptides is a variant that retains a functional capability of the parent polypeptide. As used herein, a "conservative amino acid

substitution" refers to an amino acid substitution that does not alter the relative charge or size characteristics of the polypeptide in which the amino acid substitution is made. Conservative substitutions of amino acids may, in some embodiments of the invention, include

substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. Polypeptide variants can be prepared according to methods for altering polypeptide sequence and known to one of ordinary skill in the art such. Non-limiting examples of functional variants of polypeptides for use daisy chain gene drives of the invention are functional variants of a Cas9 polypeptide, functional variants of detectable label sequences, etc.

As used herein the term "variant" in reference to a polynucleotide or polypeptide sequence refers to a change of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleic acids or amino acids, respectively, in the sequence as compared to the corresponding parent sequence. For example, though not intended to be limiting, a variant guide RNA sequence may be identical to that of its parent guide RNA sequence except that 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleic acid substitutions, deletions, insertions, or combinations thereof, and thus is a variant of the parent guide RNA. In another non-limiting example, the amino acid sequence of a variant Cas9 nuclease polypeptide may be identical to that of its parent Cas9 nuclease except that it has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions, deletions, insertions, or combinations thereof, and thus is a variant of the parent Cas9 nuclease. Certain methods of the invention for designing and constructing daisy chain gene drives include methods to prepare functional variants of daisy chain gene drive components such as guide nucleic acids, guide RNAs, and guide DNAs. Methods provided herein, and other art-known methods can be used to prepare candidate guide sequences that can be tested for function and to determine whether they retain sufficient activity for use in a daisy chain gene drive of the invention.

Methods of the invention provide means to test for activity and function of variant sequences and to determine whether a variant is a functional variant and is suitable for inclusion in a daisy chain gene drive of the invention. Suitability can, in some aspects of methods of the invention, be based on one or more characteristics such as: expression; cell localization; gene-cutting activity, efficacy in modulating activity of a target gene, etc.

Functional variant polypeptides and functional variant polynucleotides that may be used in daisy chain gene drives of the invention may be amino acid and nucleic acid sequences that have at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to their parent amino acid or nucleic acid sequence, respectively.

Art-known methods can be used to assess relative sequence identity between two amino acid or nucleic acid sequences. For example, two sequences may be aligned for optimal comparison purposes, and the amino acid residues or nucleic acids at corresponding positions can be compared. When a position in one sequence is occupied by the same amino acid residue, or nucleic acid as the corresponding position in the other sequence, then the molecules have identity/similarity at that position. The percent identity or percent similarity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity or % similarity = number of identical positions/total number of positions x 100). Such an alignment can be performed using any one of a number of well- known computer algorithms designed and used in the art for such a purpose. It will be understood that a variant polypeptide or polynucleotide sequence may be shorter or longer than their parent polypeptide and polynucleotide sequence, respectively. The term "identity" as used herein in reference to comparisons between sequences may also be referred to as "homology".

Preparation and Delivery Components of daisy chain gene drives of the invention may be delivered into a cell using standard molecular biology techniques. In certain aspects of the invention, vectors are used to implement a daisy chain gene drive of the invention, for example, to deliver a daisy chain gene drive element to a cell. As used herein, the term "vector" used in reference to delivery of components of a daisy chain gene drive system refers to a polynucleotide molecule capable of transporting between different genetic environments another nucleic acid to which it has been operatively linked. One type of vector is an episome, i.e., a nucleic acid molecule capable of extra-chromosomal replication. Some useful vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked.

Vectors capable of directing the expression of genes to which they are operatively linked may be referred to herein as "expression vectors". Other useful vectors, include, but are not limited to viruses such as lentiviruses, retroviruses, adenoviruses, and phages. Vectors useful in some methods of the invention can genetically insert one or more of a gene drive cassette into a dividing or a non-dividing cell and can insert one or more daisy chain gene drive elements into an in vivo or in vitro cell.

Vectors useful in methods of the invention may include sequences including, but not limited to one or more promoter sequences, enhancer sequences, 3' untranslated region (3'UTR) sequences, guide nucleic acid sequences, guide RNA sequences, DNA binding protein encoding sequences, detectable label encoding sequences, etc. Methods of the invention can be used to design and construct vectors comprising components of daisy chain gene drive systems. Expression vectors and methods of their use are well known in the art.

Promoters that may be used in methods and vectors of the invention include, but are not limited to, cell-specific promoters or general promoters. Methods for selecting and using cell-specific promoters and general promoters are well known in the art.

Hosts, Cells, Cell Lines, and Organisms

One or more methods of the invention for designing and constructing daisy chain gene drives as described here can be applied to prepare and deliver a daisy chain gene drive into a host cell or organism. A host cell or organism is one to which a daisy chain gene drive is delivered. In some aspects of the invention, a host cell and its progeny are understood to be member of a cell strain that includes the daisy chain gene drive, and may be referred to as daisy gene drive strain or a daisy drive strain. Similarly, a host organism and its progeny that include a daisy chain gene drive designed or prepared using one or more methods of the invention, may be referred to as an organism of a daisy drive strain, or daisy chain gene drive strain organisms, or simply as a daisy drive strain. A mutant lineage of an organism that is prepared using a daisy chain gene drive may be also be referred to as a "strain".

Daisy chain gene drive systems may be delivered to cells and organisms at various developmental stages of the cells and organisms, respectively. Non-limiting examples of stages of cells to which a daisy chain gene drive system of the invention may be delivered or included are: embryonic cells, germline cells, gametes, cells that can give rise to a gamete, zygotes, pre-meiotic cells, post-meiotic cells, fully-differentiated cells, and mature cells. Cells at this stages may be isolated cells, cells in cell lines, cells in cell, tissue, or organ culture, cells that are within an organism. In certain embodiments of the invention, a cell is a zygote, a gamete, a cell that is able to give rise to a gamete, a germline cell, etc.

Daisy chain gene drive systems designed and constructed using one or more methods of the invention may be delivered to and included in cells of various organisms. In some aspects of the invention, a cell or organism is a vertebrate or an invertebrate cell or organism. In certain aspects of the invention, a cell or organism is a eukaryotic or prokaryotic cell or organism. Non-limiting examples of organisms to which a daisy chain gene drive designed using one or more methods of the invention may be delivered to or included in are: insects, fish, reptiles, amphibians, mammals, birds, protozoa, annelids, mollusks, echinoderms, flatworms, coelenterates, and arthropods, including arachnids, crustaceans, insects, and myriapods. In some aspects of the invention an organism selected for inclusion of a daisy chain gene drive designed and constructed is an organism selected because of a population of the organism that is of interest to control or modify. As a non-limiting example, if it is of interest to control a wild population of a species of mosquito in an area or region, one or more methods of the invention are used to design and construct a daisy chain gene drive for that specific species; the designed daisy drive gene system is delivered to and included in one or more host mosquitoes of that species; one or more of the daisy chain gene mosquito strain is released into the population of wild mosquitoes; and the release of the daisy chain gene drive mosquito strain organisms controls and modulates the local wild mosquito population.

In certain aspects of the invention, an organism species to which a daisy chain gene drive designed using one or more methods of the invention may be delivered to, or included in, is a species that serves as a vector for disease affecting humans, animals, or plants. The term "vector" as used herein in reference to disease transfer, means any organism that carries and transmits an infectious pathogen into another living organism.

Some embodiments of methods of the invention for one more of designing, constructing, implementing daisy chain gene drives and systems may also be used to design, prepare, and deliver daisy chain gene drives to plants and cells thereof, including: monocots and dicots, weeds, invasive plants, poisonous plants, aquatic plants, terrestrial plants, recombinant plants, etc. Combination with traditional population control measures

It will be understood that daisy chain gene drives designed and/or constructed using one or more methods of the invention, can be introduced into cells, cell lines, and/or organisms that are released into wild populations of organisms of the same background strain. Such releases may be used in methods to suppress a wild population of the organisms.

Population reduction using daisy chain gene drives designed using methods of the invention may be used in combination with other art-known means to reduce or control the size, range, density, etc. of a population of organisms.

A population of organisms may be a local population, non-limiting examples of which is a population in a geographically defined region, such as a forest, swamp, field, pond, island, etc. and a population in a politically defined region, such as a town, state, county, etc. For example, though not intended to be limiting: to reduce the size of a wild population of a species of mosquitoes that is a known vector for a pathogen, such as malaria, eastern equine encephalitis (EEE), etc., one more methods of the invention can be used to design, construct and/or implement a daisy chain gene drive system that when included in organisms released into the wild population, is effective to decrease the size of the mosquito population.

It will be understood that daisy chain gene drive systems of the invention can be used alone or used in any combination of: before, simultaneously with, and after use of one or more alternative methods to modulate a wild population. Non-limiting examples of alternative modulation methods include: administration of pesticides, herbicides, anti -fertility agents; habitat eradication or disruption; release of organisms predatory upon the wild population; etc. Those skilled in the art will be able to identify additional population control means and to use alternative population modulation methods in combination with daisy chain gene drive methods of the invention. Population Modulation/Control

Aspects of the invention are drawn to methods to design, construct, and deliver daisy chain gene drives into cells and organisms and the release of such organisms into wild populations to modulate and control populations of species. For example, though not intended to be limiting, a daisy chain gene drive designed, constructed, and/or prepared using one or more methods of the invention is released into a wild population of an invasive species to control or eliminate that population of the invasive species. Daisy chain gene drives described herein have particular practical utility with vector-borne diseases. Malaria, dengue, yellow fever, trypanosomiasis, leishmaniasis, Chagas disease, and Lyme disease are non- limiting examples of disease caused by pathogens that are spread using vectors. Risk to subjects from infection or illness-promoting organisms may be reduced or eliminated by reducing a wild population of the organism or a vector thereof, using one or more daisy chain gene drives designed using methods of the invention. Subjects that may be protected using daisy chain gene drives designed using one more methods of the invention include, but are not limited to: humans, domesticated animals, agricultural animals, agricultural plants, wild animals, native/wild plant etc.

Field Trials and Safeguards

Certain aspects of methods of the invention include field testing. Unlike previous global gene drive systems, methods of the invention provide designs for daisy chain gene drives that can be safely tested in field trials. Daisy drive systems, designed using methods of the invention, may be capable of mimicking the molecular effects of any given global drive on a local level, and may be powerful enough to eliminate all copies of an unwanted global drive system through local immunizing reversal or population suppression, and may be field tested. Daisy drive systems designed and constructed using methods of the invention, may provide controlled and persistent population suppression by linking a sex-specific effect to a genetic locus unique to the other sex. For example, though not intended to be limiting, female fertility genes such as those recently identified in malarial mosquitoes (Hammond, A. et al., Nat Biotechnol. 2015 Dec 7; doi: 10.1038/nbt.3439) can be targeted by a genetic load daisy drive whose basal element is located on the Y chromosome or an equivalent male- specific locus (Fig. 16). These daisy chain gene drive males would suffer no direct fitness cost due to suppression relative to competing wild-type males. Another non-limiting example is a 3-element daisy drive system wherein female fertility gene disruption occurs early in development, creating a male-linked dominant sterile-daughter effect that is otherwise difficult to generate genetically. Methods of designing and constructing daisy chain gene drives as set forth herein can be used to titrate local population levels of an organism in a controlled and reversible manner, and may be useful in activity such as modulating populations of organisms, reducing populations of detrimental organisms, and studying organisms and their ecological interactions. Evolutionary Stability and CRISPR Multiplexing

Aspects of the invention include design and construction methods that overcome previous technological limitations and permit safe use of daisy drive elements. Specifically, design and construction methods of the invention can be used to reduce or eliminate risk of a recombination event that would move one or more guide RNAs within basal element of the chain into a higher element. Such a recombination event would convert a linear daisy drive chain into a self-sustaining CRISPR gene drive 'necklace' (Fig. 5). Methods of the invention include design strategies that eliminate regions of homology between the elements. Aspects of methods of the invention include, removal of promoter homology, for example, by using different U6, HI, or tRNA promoters for each element. Various promoters are known in the art and may be used in methods of the invention, see for example, Port et al. (2014) PNAS doi: 10.1073/pnas.1405500111; Ranganathan et al (2014) Nat. Comm.

doi : 10.1038/ncomms5516; and Mefferd et al (2015) RNA doi : 10.1261/rna.051631.115, the content of each of which is incorporated herein by reference. Methods of the invention, in some aspects include design and construction of daisy chain gene drives that include multiple guide RNAs expressed from a single promoter using tRNA processing (see for example: Xie et al. (2015) PNAS doi: 10.1073/pnas.1420294112, Port and Bullock (2016) bioRxiv

10.1101/046417), the content of each of which is incorporated herein by reference) or by connecting a pair of sgRNAs by a short linker, or by employing a CRISPR system that does not require independent processing. In certain aspects of designs of the invention, each gene drive element includes guide RNAs that including targeting sequences are greater than 40 or are greater than 80 base pairs in length. Design, Construction, and Use

Methods to design and construct RNA-guided gene drives based on CRISPR/Cas9 have been developed. Use of the methods singly, in combination of two or more, and in combination of one or more with other design methods for gene drives permits daisy chain gene drives to be designed, constructed, and used. Daisy chain gene drives prepared using one or more methods described herein are included in cells, cell lines, and/or organisms.

Daisy chain gene drives designed using methods provided herein can be used to address otherwise intractable ecological problems, with a level of safety inherent in their design, that reduces or eliminates a likelihood of global effects as occurs for conventional gene drive organisms that are released into the wild. Daisy gene drive elements and systems designed and/or constructed using methods provided herein are used to reduce instances and control vector-borne and parasitic diseases such as, but not limited to:, malaria,

schistosomiasis, dengue, and Zika virus. They may also be used to control or eliminate populations of agricultural pests or invasive species.

Gene drive elements and systems designed and/or constructed using one or more methods provided herein, include molecular constraints that when included in an organism or population of organisms, limit geographic spread in a tunable manner. Gene drive design and construction methods set forth herein are used in ecological engineering by enabling local communities to make decisions concerning their own environments.

Designing and constructing KNA-guided DNA nuclease gene drive elements that target multiple sequences but do not themselves encode repetitive elements.

Methods of the invention are provided that, in some embodiments, include targeting multiple sites by identifying sets of guide RNAs with very little homology to one another.

Additionally, a set of highly active guide RNA sequences is disclosed in Fig. 6 that have been verified to function with the most commonly used CRISPR system, that of S. pyogenes. A smaller set of active guide RNA sequences is disclosed in Table 1 that have been verified to function with the AsCpfl CRISPR system, which does not require external processing elements.

Table 1. Active AsCpfl repeat variants. Sequence regions and corresponding nucleotides are shown in the first five columns. SEQ ID NOs: 35-37 are each 19 nucleotides in length and SEQ ID NOs: 38 and 39 are each 20 nucleotides long.

These can be encoded in RNA-guided CRISPR gene drive systems to promote high penetrance and evolutionary stability. Guide RNAs may be expressed using a single

Polymerase III or (less efficiently) Polymerase II promoter along with sequences promoting processing as needed, such as tRNAs, using previously described methods known to those in the art that are incorporated herein by reference (Xie et al 2015 PNAS

doi : 10.1073/pnas.1420294112 , Mefferd 2015 RNA doi : 10.126 l/rna.051631.115 , Port and Bullock bioRxiv doi: 10.1101/046417). Alternatively, Cas9 and Cpfl spacers may alternate with both nucleases expressed, causing Cpfl to process the array into pairs of active guide RNAs, one corresponding to each nuclease. Alternatively, two guide RNAs may be expressed from a single Polymerase III promoter using 5-50 base pair linkages between the two guide RNAs. Alternatively, each guide RNA may be expressed from its own promoter, which may be a Polymerase III promoter. Suitable Polymerase III promoters with minimal homology are known to those in the art, e.g. U6, HI, and tRNA promoters (Port et al 2014 PNAS doi: 10.1073/pnas.1405500111, Ranganathan et al 2015 doi: 10.1038/ncomms5516).

Design and Construction ofRNA-guided DNA nuclease gene drive elements

Methods of the invention, in part, include designing and constructing RNA-guided DNA nuclease gene drive elements that target multiple sequences within genes whose loss impairs successful gametogenesis and are active in the germline after the soma-germline division has been specified but before meiosis.

Gene drive elements spread most effectively when they are minimally costly to the organism. Targeting multiple sites within genes important for fitness can avoid creating drive-resistance alleles, but still creates a fitness cost due to the effects of losing such an important gene whenever repair occurs by the wrong mechanism (e.g. not homologous recombination).

Methods of designing and constructing gene drives in which this fitness cost is reduced or eliminated by specifically targeting and recoding genes that are not just important for fitness, but are specifically important for the successful progression of gametogenesis, e.g. the production of sperm and/or eggs, are disclosed. Any event caused by such a drive element that expressed in the germline, and in some instances in the early germline, prior to meiosis impairs the ability of the cell to progress through gametogenesis (Fig. 7). [A non- limiting example of such a gene is hnRNP-GT in the mouse (Ehrmann, I, et al., Hum Mol Genet. 2008 Sep 15; 17(18):2803-18; and others are known in the art.] Because other cells in which the drive element is correctly copied are not so impaired and compensate for the absence of the impaired cells that are lost, there is little if any loss in total gamete production, and hence little if any fitness cost to the drive element due to improper repair events. This design strategy permits efficient gene drive in organisms in which it might otherwise not be possible, and notably increase the efficacy in all others.

Building and using serially dependent 1 -dimensional daisy chains of gene drive elements (daisy drive) organisms

Methods of the invention, in part, include designing, constructing, and using serially dependent 1 -dimensional daisy chains of gene drive elements (daisy drive) organisms (N- element generic) with an arbitrary number of elements such that the terminal element exhibiting drive encodes the only RNA-guided DNA nuclease, such that any new "effector" element encoding its own guide RNAs can be added in order to alter or suppress populations, or to control the activity of the resulting drive system.

Some aspects of methods of the invention can be used to construct serially dependent CRISPR gene drive elements arranged in a daisy chain, which together form a "daisy drive" system (Fig. 8). They are arranged as a series of letters in the order opposite the alphabet, such that the terminal element is always "A"; the "A" element may be accompanied by one or more "effector" elements resulting in population modulation. Because the proximal element in the chain (e.g. C in a three-element daisy drive system) does not exhibit drive, its abundance is typically limited to the initial frequency at which it is released in the population, modulated by the fitness cost of all the daisy drive elements to the organism. The next element exhibits drive only when the proximal element is present, and so tends to lose the ability to exhibit drive swiftly (Fig. 3)

Recombination events between the elements of a linear daisy drive system have the potential to create a necklace of mutually dependent elements that can exhibit global drive (Fig. 5). Hence, homology between the elements must be minimized, which is accomplished using methods such as those set forth in: Example 1, Method 1.0 to identify highly divergent guide RNA sequences; Example 2, Method 2.3 to identify different promoters; and Method 3.1 to express multiple guide RNAs with minimal homology.

Models that were prepared predict that daisy drives are more effective (e.g. they behave more like global self-sustaining drive systems for additional generations) the more elements they have. Constructing daisy drive organisms requires that one independent gene insertion event must occur for every element in the chain. Method have been determined that can be used to generate daisy drive chains capable of different purposes, such as the alteration of distinct genes or of population suppression, using the same base chain. Specifically, as described elsewhere herein, methods have been developed for designing and constructing a daisy chain gene drive organism containing N elements, generated such that the terminal element in that chain (hereby designated the A element) encodes the RNA-guided DNA nuclease. Subsequently, a) a new effector element accomplishing the desired change, be it alteration or suppression, and also encoding guide RNAs enabling it to drive in the presence of the RNA-guided DNA nuclease, is added to the organism's genome directly by standard methods known to those in the art so as to create a complete N+effector-element daisy drive organism, or b) a new effector element accomplishing the desired change, be it alteration or suppression, and also encoding guide RNAs enabling it to drive in the presence of the RNA- guided DNA nuclease, is separately inserted into the genome of another organism of the same species, which then is crossed with the daisy drive line in the laboratory so as to create a complete N+effector-element daisy drive organism, or c) the N element organisms are released into the environment to initiate a daisy drive effect that spreads the gene encoding the RNA-guided DNA nuclease through the local population, after which organisms encoding a desired "effector" element can be subsequently released to accomplish the desired effect, noting that while suppression will eliminate the RNA-guided DNA nuclease from the population, alteration can be accomplished multiple times in series or in parallel using different effector elements.

Building and using serially dependent 1 -dimensional daisy chains of gene drive elements (daisy drive) organisms

Methods of the invention, in part, include designing, building, and using serially dependent 1 -dimensional daisy chains of gene drive elements (daisy drive) organisms wherein the terminal element that exhibits drive results in population suppression through either sex-biasing (via targeting a sex chromosome in the germline after the soma-germline division has been specified but before meiosis such that surviving gametes will produce individuals mostly of one sex) or genetic load, (via disrupting genes essential for viability or fertility in one or both sexes in the germline after the soma-germline division has been specified but before meiosis).

Methods of suppressing populations using endonuclease gene drive elements to bias the sex ratio or impose a genetic load have been previously described (Burt, A. 2003 Proc. Roy. Soc. Lond. B. 270,921-8; and Esvelt, K, et al., 2014 eLife:e03401, the content of each of which is incorporated herein by reference in its entirety). However, such gene drive elements are inherently self-sustaining and consequently pose risks to all populations of the target species anywhere in the world. New methods are provided herein that are used to limit population suppression to local rather than global populations by creating "daisy chain" gene drive elements that cause local population suppression.

Specifically, methods are provided for the design and construction of a daisy drive chain of any length can be constructed in which the each element requires the prior link in order to drive, and the first element in the chain does not exhibit drive. By including a terminal effector element that imposes genetic load (Fig. 14) or generates a sex -biasing effect, the daisy drive element suppresses the population in the area of release, but because it is a limited daisy drive rather than a self-sustaining drive, that effect will be limited to the area of release.

Any potential configuration of daisy drive elements can be adjusted using methods provided herein to induce a population suppression effect. For example, a daisy chain gene drive can be designed and constructed in which target effector element can replace and therefore eliminate a recessive gene that is important for viability or fertility as would a self- sustaining/global genetic load drive, or a daisy chain gene drive may be designed and constructed that includes multiple guide RNAs that target and disrupt such a gene.

Alternatively, element A may be a standard daisy drive element (as described in Example 3, Method 3.0) that also encodes both guide RNAs targeting such loci for disruption as well as guide RNAs causing itself to drive. Alternatively, the A element or an effector element could include an extra copy of the single gene or set of genes that ensure the organism will develop a one particular sex in the relevant specie; for example, a single copy of the Sry gene in mice causes maleness. Alternatively, the A element or an effector element could include guide RNAs inducing the RNA-guided DNA nuclease to cut and eliminate a sex chromosome, thereby ensuring that nearly all offspring of A or A+effector element organisms are of one sex. These and other strategies for population suppression can be utilized in methods to design and construct daisy chain gene drives.

Methods of the invention, in part, include designing, constructing, and using serially dependent 1 -dimensional daisy chains of gene drive elements (daisy drive) organisms with an arbitrary number of elements such that the terminal element targets and recodes a gene important for organismal fitness as it spreads in order to enable the subsequent alteration or suppression of exclusively the previously altered local population at a later date.

Altering a population with a daisy drive permits subsequent precision targeting of the introduced sequence with a global CRISPR gene drive system, which will not spread beyond the target population. This is a "precision drive" strategy. It is most effective if the "A" element or an effector element of the daisy drive alters a gene suitable for targeting with a suppression drive. Single stage, two-stage, and multiple-stage suppression daisy chain gene drive systems can be designed, constructed, and implemented using methods of the invention.

Achieving stable population suppression.

Methods of the invention, in part, include achieving more stable population suppression by locating the first element in the daisy drive chain in a position unique to one sex and suppressing fertility or viability of the other sex. Daisy drive systems of the invention used directly for population suppression may experience a fitness cost limiting their potency. It is possible to ensure that the incidence of the daisy drive remains nearly proportional to the current population by reducing the fertility or viability of one sex while locating the first element of the daisy chain adjacent to a gene unique to the other sex.

For example, a simple C- B- A daisy drive might encode the guide RNAs of the C element adjacent to a male-determining gene (for example, but not limited to: the Nix gene within the M factor of the dengue vector Aedes aegypti) or a sex chromosome unique to males (for example, but not limited to: the Y chromosome in the malaria vector Anopheles gambiae). The RNA-guided DNA nuclease is encoded at the A element as is standard for a daisy drive. The effector element would include guide RNAs that target and either disrupt or replace female fertility or viability genes. Alternatively, guide RNAs disrupting these genes might be encoded on the A element, transforming it into a hybrid A/effector and leaving the drive system without a separate effector element.

As a result, daisy drive males would inactivate the female fertility genes during gametogenesis. Their sons would always inherit the C element (as well as B and A thanks to drive) and would suffer minimal fitness penalty, allowing them to repeat the cycle as it occurred in their fathers. Daughters would inherit one copy of the B element and the A element. During gametogenesis, the A element and effector elements would exhibit drive, so all offspring of these daughters would inherit a broken copy. If the other parent is a daisy drive male, their daughters will be sterile, thereby suppressing the population.

Methods of the invention, in part, include achieving stable population suppression with a daisy intermediate designed, constructed, and used to inactivate female fertility genes in a dominant manner. A variation on the above population suppression methods involves ensuring that the A element and optionally effector elements exhibit drive in the zygote, thereby ensuring that any female inheriting a single copy is sterile (or nonviable). This is achieved by arranging for the RNA-guided DNA nuclease encoded in A to be expressed in the zygote and/or the early stages of development, or by utilizing a separate RNA-guided DNA nuclease that is so expressed. This will cause the drive system to disrupt the wild-type allele of the target gene inherited from the other parent, resulting in sterile or nonviable females. Because the fitness cost to males will be minimal, the introduction of males of this type will cause immediate population suppression proportional to the fraction of daisy drive males (Fig. 15). This approach is often necessary because there are few genes whose loss causes dominant sterility in a sex.

Another variation on the above population suppression methods is illustrated in Fig. 16. Fig. 16 illustrates a daisy drive that imposes a genetic load on female fertility as designed and constructed in Example 4, Method 4.0, but one in which the proximal element (C in this case) is embedded within a male-exclusive genetic element to mitigate the fitness cost as set forth in Example 6, Method 6.0. Rectangles highlight mating events that trigger sterility in female offspring. Designing and constructing daisy drive elements in which guide RNAs are embedded within introns of target genes.

Methods of the invention, in part, include designing, constructing, and using daisy drive elements in which guide RNAs are embedded within introns of target genes. Some genes may not be amenable to recoding at the 3' end, or to having their 3'UTR replaced. An alternative method is described in which the guide RNAs are encoded within the gene itself. This is most effective when the gene is highly transcribed; fortunately, most haploinsufficient genes chosen as daisy drive targets are ribosomal and are consequently some of the most highly expressed in the cell. However, guide RNAs must be produced from these transcripts without disrupting the function of the gene. A solution has been developed that includes embedding the guide RNAs within introns, separated by tRNAs for efficient processing, or alternatively by using guide RNAs that do not require external processing factors, or alternatively by incorporating such guide RNAs instead of tRNAs and expressing both nucleases. The tRNA-processing method has been shown to enable high nuclease activity in fruit flies when driven by strong polymerase II promoters (see: //dx.doi. org/10.1101/046417); ribozyme-based processing (not suitable for daisy drive due to repetitiveness) works efficiently from within introns (see: //dx. doi. org/10.1016/j .molcel.2014.04.022). To ensure that the guide RNAs are copied efficiently, the target wild-type gene should be cleaved on both sides of the intron. A less evolutionarily stable but still viable form of the drive system cleaves the wild-type but not the engineered intron. Building evolutionarily unstable yet robust drive systems through redundancy.

Methods of the invention, in part, include designing, constructing, and using homing- based gene drive systems that are not vulnerable to drive-resistant alleles that block drive copying and thus prevent the spread of the drive system. These alleles are generated naturally whenever the endonuclease cut is repaired by non-homologous end-joining, which can create indels or point mutations at the target site that block subsequent cutting. This is why evolutionarily stable drives target multiple sites within genes important for fitness.

The invention in part also includes methods to identify highly active guide RNA sequences that share minimal homology that may be included in a daisy chain gene drive system of the invention, and may enable evolutionary stable daisy drive as well as global CRISPR gene drive. However, it is possible to affect large numbers of organisms even without evolutionary stability. A typical rate of NHEJ repair is 5% (Gantz, V. & Bier, E. 2015 Science 24 Apr: Vol. 348, Issue 6233, pp. 442-444; Gantz, V. et al., 2015 PNAS Vol. 112 no. 49 E6736-E6743; and Hammond, A. et al., Nat Biotechnol. 2015 Dec 7;

doi: 10.1038/nbt.3439, each of which is incorporated herein by reference in its entirety). Thus, at minimum 5% of the population will be unaffected by the drive system; the share will decline as natural selection favors the resistant alleles over the drive. This precludes suppression drive strategies, but may be acceptable for certain alteration-based requirements. One method of compensating is to build multiple evolutionarily unstable drive systems, each of which targets a single site, wherein each drive system can overwrite resistance alleles generated by the others, but cannot directly overwrite one another. This multiple-drive approach is less stable than using a single drive system that targets multiple sites within a sequence important for fitness because resistance alleles could accrue one by one in the former but not the latter, and also requires building many drive systems which complicates modeling and regulation. However, there is no need to target a sequence important for fitness.

Similar logic applies to daisy drive systems. Because a daisy drive system is not intended to spread indefinitely, each element will only be copied a fixed number of times. This limits the potential for drive-resistant alleles to emerge that block spread, especially for non-A, non-effector elements. However, this is counterbalanced by the increased number of elements that must be copied, which increases vulnerability to any one drive-resistant allele. Building multiple daisy drive elements at each position, all of which can overwrite resistance alleles that block the other versions, can compensate for this deficit, as can ensuring that each daisy element targets the nuclease-encoding A element as well as the next element in the chain (Fig. 9).

The following examples are provided to illustrate specific instances of the practice of the present invention and are not intended to limit the scope of the invention. As will be apparent to one of ordinary skill in the art, the present invention will find application in a variety of compositions and methods.

EXAMPLES

Example 1

Methods to design and construct RNA-guided gene drives based on CRISPR/Cas9 have been developed. Use of the methods singly, in combination of two or more, and in combination of one or more with other design methods for gene drives permits daisy chain gene drives to be designed, constructed, and used. Daisy chain gene drives prepared using one or more methods described herein are included in cells, cell lines, and/or organisms.

Daisy chain gene drives designed using methods provided herein are used to address otherwise intractable ecological problems, with a level of safety inherent in their design, that reduces or eliminates a likelihood of global of daisy chain gene drive organisms that are released into the wild. Daisy gene drive elements and systems designed and/or constructed using methods provided herein are used to reduce instances and control vector-borne and parasitic diseases such as, but not limited to:, malaria, schistosomiasis, dengue, and Zika.

Gene drive elements and systems designed and/or constructed using one or more methods provided herein, include molecular constraints that when included in an organism or population of organisms, limit geographic spread in a tunable manner. Gene drive design and construction methods set forth herein are used in ecological engineering by enabling local communities to make decisions concerning their own environments.

Methods of designing and constructing RNA-guided DNA nuclease gene drive elements that target multiple sequences but do not themselves encode repetitive elements.

Endonuclease gene drive systems continually create alleles that they cannot replace whenever nuclease-cut DNA is repaired by non-homologous or microhomology-mediated end-joining or a similar pathway in a manner that mutates the recognition site of the endonuclease. If the resulting mutant allele confers higher fitness to the organism than the drive system, natural selection will favor the mutant drive-resistant allele, preventing the drive system from ever reaching fixation and eventually leading to its elimination from the population. Targeting a gene important for fitness can reduce the frequency at which this occurs, but synonymous mutations or non-synonymous mutations, in-frame insertions, or deletions could still preserve function and outcompete the drive system.

A reliable method of overcoming this problem is to program the endonuclease to cut multiple nearby sites within a gene important for fitness such that any repair method that does not involve homologous recombination (and hence copying of the drive system) deletes the portion of the gene between the cut sites and consequently creates a loss-of-function mutation that is more costly than the drive (Esvelt et al 2014 //dx.doi. org/10.7554/eLife.03401).

Targeting multiple sites also reduces the chance of each cut being repaired to create a minimally costly mutation independently; the more sites targeted, the lower the chance of any allele acquiring resistance to each cut. However, this multi-site targeting must be

accomplished without introducing repetitive sequences into the drive system, as internal repeats frequently lead to internal recombination and instability of the drive cassette(Simoni et al Nucleic Acids Research 2014 //dx. doi.org/10.1093/nar/gku387). Such an event could inactivate the drive system, which reduces its overall efficiency, or worse yet might reduce or eliminate its ability to target multiple sites, thereby promoting the emergence of resistance alleles which in turn could lead to the serial acquisition of resistance to all cut sites.

CRISPR systems can readily target multiple sites using different guide RNAs, but each of these must be separately encoded in a way that does not permit internal

recombination.

Methods are provided that enable targeting multiple sites by identifying sets of guide RNAs with very little homology to one another. Additionally, a set of highly active guide RNA sequences is disclosed that have been verified to function with the most commonly used CRISPR system, that of S. pyogenes. These can be encoded in RNA-guided CRISPR gene drive systems to promote high penetrance and evolutionary stability. Guide RNAs are expressed using a single Polymerase III or (less efficiently) Polymerase II promoter along with sequences promoting processing, such as tRNAs, using previously described methods known to those in the art that are incorporated herein by reference (Xie et al 2015 PNAS doi: 10.1073/pnas.1420294112, Mefferd 2015 RNA doi: 10.1261/rna.051631.115, Port and Bullock bioRxiv doi: 10.1101/046417). Alternatively, two are expressed from a single Polymerase III promoter using 5-50 base pair linkages between the two guide RNAs. Finally, each guide RNA is expressed from its own promoter, which may be a Polymerase III promoter. Suitable Polymerase III promoters with minimal homology are known to those in the art, e.g. U6, HI, and tRNA promoters (Port et al 2014 PNAS

://dx.doi. org/10.1073/pnas.1405500111). Alternatively, guide RNAs from a CRISPR system that does not require external processing, such as one employing Cpfl, may be used; a set of mutant guide RNAs for AsCpf 1 are shown in Table 1. Alternatively, these guide RNAs may be placed in an alternating array with guide RNAs that do not display this activity and both nucleases expressed, causing processing of the array into pairs of active guide RNAs.

A problem may arise because of the length of the portion of the guide RNA sequence that is recognized by the CRISPR system, which may be Cas9 from S. pyogenes. This portion is over 60bp in length, which is more than enough for internal recombination (Mali et al 2013 //dx.doi.org/10.1126/science.1232033). Recombination was identified as undesirable in gene drives.

The known crystal structure (Nishimasu et al 2014 Cell

//dx.doi. org/10.1016/j .cell.2014.02.001) and data on guide RNAs from closely related CRISPR systems (and synthetic variants) (Briner 2014 Molecular Cell

//dx.doi. org/10.1016/j .molcel.2014.09.019) that can be recognized by S. pyogenes Cas9 was used to identify candidate regions thought to be more or less important for guide RNA recognition. A set of guide RNAs was prepared using Method 1.0 (Fig. 2) and their activity was measured their activity to the "wild-type" sgRNA using a transcriptional activation assay (Fig. 3) (Mali et al 2013 Nature Biotechnology //dx.doi.org/10.1038/nbt.2675).

A similar set of guide RNAs with minimal homology is created for any given

CRISPR system through equivalent means. If nothing is initially known of the relevant dependencies, the relevant information is gleaned by performing structural studies similar to those referenced, or through a library-based approach (Method 1.1) followed by design and assaying (Method 1.0). Method 1.0 permits rapid preparation of DNA sequences capable of expressing multiple guide RNAs. Methods of the invention permit rapid identification and preparation of repetitive sequences, which was not previously possible.

Method 1.0 - creating highly divergent guide RNA variants with minimal homology to one another.

(1) All known relevant information was used to create a list or map of the guide RNA denoting every possible individually accepted change throughout the structure. If there is insufficient information, Method 1.1 can be used to generate the relevant dataset.

(2) Several dozen elements were designed that combined permutations of these permitted changes so as to minimize the length of sequences shared between elements. In this context, the term "elements" means the backbone of the guide RNA sequence recognized by the nuclease that is capable of directing the nuclease to cut a target sequence.

(3) Activity of these designed sequences was measured e.g. by a transcriptional activity reporter assay as detailed below, or using a selection method as detailed in Method 1.2.

(4) All divergent guide RNA sequences that retained high activity were identified and recorded. These divergent guide RNA sequences are suitable for, and are used for, constructing evolutionarily stable homing-based gene drive systems that target multiple sites to overcome the evolution of mutations that block cutting. Because the divergent sequences dramatically reduce the chance of recombination between homologous sequences within the drive cassette, which is a major problem for highly repetitive drive cassettes (Simoni et al Nucl. Acids Res. 2014 //dx. doi.org/10.1093/nar/gku387), the resulting drive systems are stable. They are also used for the construction of functional daisy drive system homology between elements to avoid recombination events that could lead to global drive activity, as detailed in Example 3, Method 3.0. These guide RNA sequences are also useful for synthesizing DNA sequences encoding multiple guide RNAs for standard multiplexing experiments involving CRISPR gene editing and regulation.

(5) Figs. 6 and 14 detail a set of highly divergent guide RNAs that were designed and prepared and indicates their activity relative to the most commonly used guide RNA for the RNA-guided DNA nuclease Cas9 from S. pyogenes. Activity was determined using the fluorescent reporter assay detailed below Method 1.1. It has previously been very difficult to synthesize repetitive sequences, which has precluded attempts to quickly make DNA sequences capable of expressing multiple guide RNAs. Methods of the invention permit rapid identification and preparation of repetitive sequences that are used in daisy chain gene drives and other gene drives.

The method was additionally utilized to identify guide RNA variants corresponding to the Cpfl nuclease system from Acidaminococcus (AsCpfl). In this case, activity was measured using a plasmid exclusion assay. Arrays of ten AsCpfl guide RNAs targeting ten different plasmids were constructed, with each guide RNA harboring different candidate mutations. Each array was expressed in an E. coli cell along with AsCpfl . These cells were transformed with plasmids encoding sequences targeted by the array and transformation efficacy compared to control cells lacking the array. A difference of >100-fold indicated functional exclusion and AsCpfl activity. Because the processing and DNA-cutting activities of AsCpfl are distinct, putatively active crRNAs were employed in additional arrays in which they were paired with different adjacent crRNAs and a different target sequence, and the assay repeated.

Method 1.1 - library-based guide RNA interrogation

(1) Two libraries are created. One is a randomized library of guide RNA sequences averaging 1-5 mutations per member and the second is a targeted library in which the base pairs in predicted hairpins are replaced with alternative base pairs that preserve the predicted hairpin structure (e.g. G-C pairs are replaced by C-G, A-T, T-A, G-T, and T-G) or create a mispair (e.g. C-C). These libraries can be generated by methods known to those in the art or synthesized as oligonucleotides by known commercial suppliers (e.g. CustomArray).

(2) Using a plasmid in which the protospacer sequence targeted by the spacer in the guide RNAs is directly adjacent to the sequence encoding the guide RNAs such that activity leads to cutting of the plasmid, transform bacteria or transfect eukaryotic cells that also express either active or inactive Cas9, perform high-throughput sequencing (such as but not limited to: Ulumina MiSeq or HiSeq methods) of the plasmid sequences encoding the guide RNAs, and identify the most active variants as those most thoroughly depleted when Cas9 is active (e.g. using method of Esvelt et al 2013 Nature Methods //dx.doi. org/10.1038/nmeth.2681).

(3) Alternatively, two protospacer sites are encoded, and the region containing both as well as the guide RNA is PCR-amplified, the resulting amplicons are size-selected for those lacking the sequence between the protospacers, and are sequenced to identify those active enough to cut both sites. See Example 2, Method 2.3.

(4) Alternatively, transcriptional activation assay with a fluorescent reporter is used as is detailed in Method 1.2 and fluorescence-assisted cell sorting is used the guide RNAs that result in the highest levels of transcriptional activation are identified.

(5) All instances in which substituting bases in a hairpin retains activity are noted.

(6) All instances in which mutating a single base preserves activity are noted.

(7) All instances in which adding or removing a base preserves activity are noted.

Method 1.2 Measuring guide RNA activity via transcriptional activation reporter assay Methods to measure and determine activity of candidate guide RNAs were designed and tested.

(1) Cells are grown using standard conditions (for example, HEK293T cells were grown in Dulbecco's Modified Eagle Medium (Life Technologies) fortified with 10% FBS (Life Technologies) and Penicillin/Streptomycin (Life Technologies), incubated at a constant temperature of 37°C with 5% C0 ₂ ).

(2) The cells were split into multi-well plates, divided into approximately 50,000 cells per well and then transfected with plasmids encoding:

(a) dCas9-VPR or an equivalent dead-nuclease transcriptional activator variant of the

RNA-guided DNA-binding protein nuclease matching the candidate guide RNAs to be tested,

(b) the candidate guide RNA to be evaluated,

(c) a reporter plasmid comprising a minimal promoter and one or more protospacer binding site upstream of a gene encoding a fluorescent protein, and

(d) a control plasmid expressing a different fluorescent marker gene as a transfection control marker.

(3) The transfections were carried out using standard methods, (for example, using 2μ1 of Lipofectamine 2000 (Life Technologies) with 200ng of dCas9 activator plasmid, 25ng of guide RNA plasmid, 60ng of reporter plasmid and 25ng of EBFP2 expressing plasmid. The reporter plasmid was a modified version of addgene plasmid #47320, a reporter expressing a tdTomato fluorescent protein adapted to contain an additional gRNA binding site lOObp upstream of the original site, the activator is da tripartite transcriptional activator fused to the C-terminus of nuclease-null Streptococcus pyogenes Cas9).

(4) After transfection, the cells were analyzed using flow cytometry to measure activity, and any cells that didn't fluoresce due to the presence of the transfection control marker were ignored.

(5) Optionally, if a library of guide RNAs is assayed at the same time, fluorescent- assisted cell sorting (FACS) is used to sort for plasmids encoding highly active guide RNAs which are then sequenced to identify.

Example 2

Methods for designing and constructing RNA-guided DNA nuclease gene drive elements that target multiple sequences within genes whose loss impairs successful gametogenesis and are active in the germline after the soma-germline division has been specified but before meiosis.

Gene drive elements spread most effectively when they are minimally costly to the organism. Targeting multiple sites within genes important for fitness can avoid creating drive-resistance alleles, but still creates a fitness cost due to the effects of losing such an important gene whenever repair occurs by the wrong mechanism (e.g. not homologous recombination). Previous studies have proposed and more recently demonstrated, or at least attempted to demonstrate, population suppression drive elements that are not active in the embryo or the soma, only in the germline (Burt, A. 2003 Proc. Roy. Soc. Lond. B. 270,921-8; Hammond et al 2015 Nature Biotech).

Methods of designing and constructing gene drives in which this fitness cost is reduced or eliminated by specifically targeting and recoding genes that are not just important for fitness, but are specifically important for the successful progression of gametogenesis, e.g. the production of sperm and/or eggs. Any event caused by such a drive element that expressed in the germline, and in some instances in the early germline, prior to meiosis impairs the ability of the cell to progress through gametogenesis (Fig. 7). [A non-limiting example of such a gene is hnRNP-GT in the mouse (doi: 10.1093/hmg/ddnl79); and others are known in the art.] Because other cells in which the drive element is correctly copied are not so impaired and compensate for the absence of the impaired cells that are lot, there is little if any loss in total gamete production, and hence little if any fitness cost to the drive element due to improper repair events. This design strategy permits efficient gene drive in organisms in which it might otherwise not be possible, and notably increase the efficacy in all others.

Method 2.0 - building evolutionarily stable gene drive systems with minimal fitness cost.

(1) A gene is chosen that is known to be haploinsufficient for normal cell growth, e.g. one wherein a single copy is insufficient for normal growth and division.

(2) If no such gene is known: a gene is chosen that encodes a ribosomal protein, most of which are haploinsufficient. Assays are performed for haploinsufficiency in the germline via Method 2.1 below as needed.

(3) A gene is identified that is first expressed exclusively in the germline after soma- germline differentiation in the organism of interest. Assays are performed for expression timing via Method 2.2.

(4) The identified gene's promoter/enhancer/3'UTR is used to drive expression of the RNA-guided DNA-binding protein nuclease (e.g. Cas9 or equivalent) in a gene drive cassette, e.g. one that also encodes guide RNAs targeting the equivalent wild-type locus, where the guide RNAs are expressed from a promoter such as one identified using Method 2.3. Optionally, the nuclease is fused to a fluorescent protein (e.g. GFP) using 2A peptide tag and use fluorescent imaging of the embryo and it is verified that expression is germline- specific and occurs at the correct developmental stage.

(5) Measurement is performed to determine lifetime fertility of organisms encoding the candidate drive cassette when mated to wild-type partners as compared to wild-type / wild- type pairings to verify that there is no loss of reproductive fitness. Offspring are screened by PCR to identify any heterozygotes in which the drive has not been copied. Method 2.1 - assaying a gene for haploinsufficiency in the germline.

(1) A strain of transgenic organisms is created in which an RNA-guided DNA-binding protein nuclease is expressed exclusively in the germline after soma-germline differentiation (see Methods 2.0, 2.2).

(2) One or more strains of transgenic organisms are created in which a single guide RNA targeting the coding region of the candidate haploinsufficient gene is expressed under a polymerase III (e.g. U6) promoter, which in some cases is one identified using Method 2.3.

(3) The two strains are crossed to create a heterozygous line in which the target gene is cut in germline cells just after soma-germline differentiation.

(4) The resulting hybrids are mated to wild-type organisms.

(5) The candidate haploinsufficient genes in the offspring zygotes or embryos, or the gametes of the original organism, are sequenced. If the gene is in fact haploinsufficient in the germline, all offspring or gametes should have intact copies resulting from cells in which the nuclease did not cut or copies with mutations that do not significantly impair the function of the gene.

Method 2.2 - expressing genes exclusively in the germline after soma-germline

differentiation.

(1) If the organism is amenable, embryos are dissected to isolate germline cells and a full transcriptome sequencing analysis is performed. Candidate genes are chosen that are identified as expressed exclusively in the germline after the soma-germline differentiation step.

(2) If transcriptome analysis is not possible, the method of Merritt et al (2008) Current Biology (10.1016/j .cub.2008.08.013) is carried out and promoters/enhancers/3'UTRs are tested for appropriate expression.

Method 2.3 - identifying highly active promoters for guide RNA expression.

(1) Into an organism or cell line expressing an RNA-guided DNA nuclease, DNA encoding one of a number of candidate promoters driving a guide RNA is delivered. This guide RNA should target one or ideally two sequences located just upstream of the promoter. (2) DNA is extracted and purified and PCR used to amplify the target site(s) as well as the candidate promoter.

(3) If using two target sites, amplicons are size-select to those lacking the sequence between the sites are identified.

(4) Sequence amplicons to identify which candidate promoters most often cleaved the target site(s), causing a mutation and/or deletion via non-homologous end-joining.

(5) Alternatively, the DNA is delivered into cultured cells of the target organism. The repeated sequences are positioned in such a way as to disrupt production of a fluorescent protein encoded on the same fragment. A second fluorescent protein is encoded as a marker for cells that have taken up the DNA. Fluorescence-assisted cell sorting (FACS) is used to enrich for cells expressing the second fluorescent protein but not the first one, indicative of successful cutting. Sequencing is performed and the most active promoters identified.

Example 3

Provided are methods of building and using serially dependent 1 -dimensional daisy chains of gene drive elements (daisy drive) organisms with an arbitrary number of elements such that the terminal element exhibiting drive encodes the only RNA-guided DNA nuclease such that any new effector element encoding its own guide RNAs can be trivially added in order to alter or suppress populations, or to control the activity of the resulting drive system.

The self-propagating nature of global gene drive renders the technology uniquely suited to addressing large-scale ecological problems, but tremendously complicates discussions of whether and how to proceed with any given intervention. Technologies capable of unilaterally altering the shared environment require broad public support. Hence, ethical gene drive research and development must be guided by affected communities and nations to an extent unprecedented in the history of science. Attaining this level of engagement and informed consent becomes more challenging as the number of people affected grows.

A way to confine the spread of a gene drive element to local populations would greatly simplify community-directed development and deployment. Prior strategies (see for example: Gould et al 2008 Proc Roy Soc B doi: 10.1111/j . l558-5646.2007.00298.x, Rasgon PLoS One 2012 doi: 10.1371/journal. pone.0005833) can locally spread cargo genes nearly to fixation if sufficient organisms (>30% of the local population) are released. "Threshold- dependent" gene drives such as those employing under-dominance (Curtis Nature 218:268- 269 1968, Akbari and Hay Curr. Biol. 2013, Reeves et al PLoS One 2014) will spread to fixation in small and geographically isolated subpopulations if enough organisms are released to exceed the threshold (typically -50%) for population takeover. These prior containment methods are not sufficiently effective or workable for use in populations in the wild.

A solution that has been undertaken is to construct serially dependent CRISPR gene drive elements arranged in a daisy chain, which together form a "daisy drive" system (Fig. 8). They are arranged as a series of letters in the order opposite the alphabet, such that the terminal element carrying the nuclease gene is always "A". Because the basal element in the chain (e.g. C in a three-element daisy drive system) does not exhibit drive, its abundance is typically limited to the initial frequency at which it is released in the population, modulated by the fitness cost of all the daisy drive elements to the organism. The next element exhibits drive only when the basal element is present, and so tends to lose the ability to exhibit drive upon loss of the basal element (Fig. 9).

Recombination events between the elements of a linear daisy drive system have the potential to create a necklace of mutually dependent elements that can exhibit global drive (Fig. 5). Hence, homology between the elements must be minimized, which is accomplished using methods such as those set forth in: Example 1, Method 1.0 to identify highly divergent guide RNA sequences; Example 2, Method 2.3 to identify different promoters; and Method 3.1 to express multiple guide RNAs with minimal homology.

Models predict that daisy drives are more effective (e.g. they behave more like global self-sustaining drive systems) the more elements they have. Constructing daisy drive organisms requires that one independent gene insertion event must occur for every element in the chain. Methods have been determined that can be used to generate daisy drive chains capable of different purposes, such as the alteration of distinct genes or of population suppression, using the same base chain. Specifically, methods have been developed for designing and constructing a daisy chain gene drive organism containing N elements such that the terminal element in that chain (hereby designated the A element) encodes the RNA- guided DNA nuclease. Subsequently, a) a new effector element accomplishing the desired change, be it alteration or suppression, and also encoding guide RNAs enabling it to drive in the presence of the RNA-guided DNA nuclease, is added to the organism's genome directly by standard methods known to those in the art so as to create a complete N+effector-element daisy drive organism, or b) a new effector element accomplishing the desired change, be it alteration or suppression, and also encoding guide RNAs enabling it to drive in the presence of the RNA-guided DNA nuclease, is separately inserted into the genome of another organism of the same species, which then is crossed with the daisy drive line in the laboratory so as to create a complete N+effector-element daisy drive organism, or c) the N-element organisms are released into the environment to initiate a daisy drive effect that spreads the gene encoding the RNA-guided DNA nuclease through the local population, after which organisms encoding any desired "effector" element can be subsequently released to accomplish the desired effect, noting that while suppression will eliminate the RNA-guided DNA nuclease from the population, alteration can be accomplished multiple times in series or in parallel using different effector elements.

There are two different Method 3.0 - Constructing a generic daisy drive organism of N elements in which each element is evolutionarily stable.

(1) A sufficient number of sequence-divergent guide RNAs are identified (e.g. using Method 1.0) for the desired number of daisy drive elements. Each element except one requires at least one guide RNA, but it is best if each encodes 2 or preferably more guide RNAs. All guide RNAs encoded in the organism are sequence-divergent to minimize the potential for recombination within and between different elements.

(2) Up to N-l target genes and expression conditions for the RNA-guided DNA nuclease (e.g. using Example 2, Method 2.0) are identified. Methods below describe designing and constructing a four-element daisy chain gene drive, but the methods are also used to create longer and shorter daisy chain gene drives, that have three elements (a C-B-A daisy chain gene drive) or five, six, seven, eight, nine, ten, or more elements in longer daisy chain gene drives.

(3) The positions of the final daisy chain elements are defined in descending alphabetical order such that position A encodes the nuclease, so, for example, a 4-element drive is D-C-B- A. In this case, this method involves creating C-B-A such that any effector element can be added. Element A is constructed first with the selection of a target gene and recoding the target gene sequence according to Method 3.3. A RNA-guided DNA nuclease is encoded downstream of the 3'UTR under appropriate expression conditions according to Example 2, Method 2.0.

(4) The B element is constructed next by encoding one or more guide RNAs recognizing the target gene of the A element just downstream of the C element target gene. The C element target gene is selected and recoded and its 3'UTR is replaced with one from another gene that has similar expression conditions (see Method 3.3). This is done in the strain containing B element or in a separate strain. The guide RNAs are designed and they are expressed using appropriate promoter(s) and processing methods - see Method 3.1. Also see Method 9.0 for an alternative way to encode guide RNAs in daisy drive elements.

(5) The elements for C, D, E, etc. are constructed as described for element B (step 4) until all the desired drive elements have been constructed. If the drive elements are constructed in separate strains, crosses are performed to combine all elements in a single strain, a process that can be assisted via the activity of the daisy drive. If a potential application for the prepared daisy chain gene drive may involve organism population suppression via a sex- specific effect, it can be advantageous to encode the highest/proximal element of the daisy chain (e.g. E in an E-D-C-B-A chain) within a locus exclusive to the unaffected sex.

(6) The resulting strain designed and constructed as in Method 3.0 exhibits daisy drive to spread element A, which encodes the RNA-guiding DNA-binding nuclease, through the local population.

(7) To add an effector element corresponding to a desired population modulation effect, another strain is made that includes the desired genomic change and guide RNAs capable of driving that change by cutting the wild-type version. To be evolutionarily stable, this element should target an important sequence at multiple sites and either recode it or replace it to accomplish the desired effect. See Example 4, Method 4.0 for additional details regarding population suppression methods. Method 3.0.1 - Constructing a generic daisy drive organism of N elements in which no element is evolutionarily stable

(1) A sufficient number of sequence-divergent guide RNAs are identified (e.g. using Method 1.0) for the desired number of daisy drive elements.

(2) Expression conditions for the RNA-guided DNA nuclease (e.g. using Example 2, Method 2.0) are identified.

(3) Element A is constructed first, and replaces a target sequence, ideally one that is reasonably conserved but not important for fitness, often called a "neutral sequence" or "neutral site" by those in the art. For example, a sequence adjacent to but not included within an essential gene's regulatory signals is often suitable for replacement; ribosomal genes are particularly useful for this. An example of a presumed neutral locus is ROSA26 in the mouse.

(4) The B element is constructed next, and replaces a wild-type sequence with a synthetic sequence encoding one or more guide RNAs that target the wild-type sequence replaced by the A element. Again, the replaced sequence is ideally conserved but not important for fitness. This is done in the strain containing the A element or in a separate strain. The guide RNAs are designed and are expressed using appropriate promoter(s) and processing methods - see Method 3.1. Also see Method 9.0 for an alternative way to encode guide RNAs in daisy drive elements.

(5) The elements for C,D, E, etc. are constructed as described for element B (step 4) until all the desired drive elements have been constructed. If the drive elements are constructed in separate strains, crosses are performed to combine all elements in a single strain, a process that can be assisted via the activity of the daisy drive.

(6) The resulting strain designed and constructed as in Method 3.0 exhibits daisy drive to spread element B, which encodes the RNA-guiding DNA-binding nuclease, through the local population.

(7) To add an effector element corresponding to a desired population modulation effect, another strain is made that includes the desired genomic change and guide RNAs capable of driving that change by cutting the wild-type version. Method 3.0.2 - Constructing a generic daisy drive organism of N elements in which only the "A " element is evolutionarily stable.

(1) A sufficient number of sequence-divergent guide RNAs are identified (e.g. using Method 1.0) for the desired number of daisy drive elements.

(2) A target gene or sequence important for organismal fitness is identified. Examples include probable haploinsufficient genes such as ribosomal genes. Expression conditions for the RNA-guided DNA nuclease (e.g. using Example 2, Method 2.0) are identified. The target gene may be the same gene that displays appropriate expression conditions; for example, Nanos3 both exhibits germline-specific expression after germline lineage specification and is essential to generate viable gametes.

(3) Element A is constructed first. It recodes the target gene or sequence important for fitness to remove the sequences to be targeted by element B while preserving the function of the gene. If the target gene offers suitable expression conditions, it is fused to the nuclease- encoding sequence using a 2A peptide so that expression of the two is transcriptionally and translationally coupled. If not, the target gene's 3'UTR is replaced with the 3'UTR of a closely related gene and the nuclease gene is inserted downstream along with an appropriate enhancer/promoter/3'UTR corresponding to the desired expression conditions.

(4) The B element is constructed next, replacing a wild-type sequence with a synthetic sequence encoding one or more guide RNAs that recognize the wild-type sequence replaced by the A element. The replaced sequence is ideally conserved but not important for fitness. This is done in the strain containing the A element or in a separate strain. The guide RNAs are designed and are expressed using appropriate promoter(s) and processing methods - see Method 3.1. Also see Method 9.0 for an alternative way to encode guide RNAs in daisy drive elements.

(5) The elements for C, D, E, etc. are constructed as described for element B (step 4) until all the desired drive elements have been constructed. If the drive elements are constructed in separate strains, crosses are performed to combine all elements in a single strain, a process that can be assisted via the activity of the daisy drive.

(6) The resulting strain designed and constructed as in Method 3.0 exhibits daisy drive to spread element A, which encodes the RNA-guiding DNA-binding nuclease, through the local population.

(7) To add an effector element corresponding to desired population modulation effect, another strain is made that includes the desired genomic change and guide RNAs capable of driving that change by cutting the wild-type version. See Example 4, Method 4.0 for additional details regarding population suppression methods.

Method 3.1 - expressing multiple guide RNAs with minimal homology

(1) If testing or previous studies of RNA interference or CRISPR-mediated genome editing have identified polymerase III promoters capable of strong RNAi or guide RNA expression, those are used in the design and construction of daisy chain gene drives.

Examples of suitable polymerase III promoters are for example: U6, HI, and tRNA promoters. If suitable promoters are not known, Example 2, Method 2.3 is used to identify promoters suitable for the type of daisy chain gene drive system that is designed and constructed. In some organisms, it may be possible to express guide RNAs from polymerase II promoters, sometimes using ribozymes or tRNAs for appropriate processing (see Method 3.2). Note that promoters cannot be re-used across daisy drive elements.

(2) It is possible to express two guide RNAs from a single polymerase III promoter by replacing the poly-T stretch leading to transcriptional termination of the first guide RNA with a 10-15 base pair linker to the second guide RNA. If earlier steps identify sufficient active promoters to express enough guide RNAs (ideally 2+ per element) for the desired daisy drive system, do so.

(3) To express more guide RNAs from a single promoter, a tRNA-based processing strategy is used. This approach also permits the guide RNAs to be processed to any desired length, potentially increasing specificity. See Method 3.2 to identify tRNAs suitable for processing. Alternatively, a self-processing CRISPR system such as Cpfl may be used, in which case a single promoter can drive numerous crRNAs in series.

Method 3.2 - Identifying tRNAs suitable for tRNA-guide RNA-tRNA array processing (1) A strain is constructed in which the RNA-guided DNA nuclease is expressed using a housekeeping gene enhancer/prom oter/3'UTR such as actin that also expresses a fluorescent protein, either from a separate promoter or via 2A peptide fusion.

(2) Additional strains are constructed in which a promoter previously demonstrated to be effective in that organism (e.g. U6/Hl/tRNA or one identified via Example 2, Method 2.3) drives a construct consisting of a tRNA, a control guide RNA that does not target any sequence in the cell, a different tRNA to be tested, a guide RNA targeting the gene encoding the fluorescent protein (or an equivalent recessive marker gene), a third tRNA, and another control guide RNA. The strains are crossed and fluorescence is measured in the progeny. Less fluorescence indicates more effective tRNA processing. The process is repeated, varying different tRNAs in the second and third positions, until sufficient tRNAs have been identified for processing of all daisy drive elements.

(3) Alternatively, the above experiment design and construction is performed in cultured cells. For example, the two DNA fragments described in the preceding paragraph are combined into one construct (which also encodes a different fluorescent protein as a marker of successful DNA delivery) and that DNA sequence is delivered into cultured cells of the target species. A standard method such as fluorescent-assisted cell sorting is used to isolate cells with the fluorescent marker that received the DNA. The cells are further sorted to identify cells that also lack the fluorescent gene targeted by the guide RNA, as these are cells in which tRNA-processing was effective in that it produced an active guide RNA that cut the fluorescent gene. The DNA is extracted and sequenced (in some instances using high- throughput) and tRNAs that worked are identified.

(4) Alternatively, a large library is prepared that includes DNA fragments encoding: (RNA-guided DNA nuclease, promoter-sitel-tRNAl-(guide RNA targeting site l)-tRNA2- (guide RNA targeting site 2)-tRN A3 -(control guide RNA)-(site 2) for many different tRNAs of interest in different combinations. These DNA fragments are delivered into cells of the target species by standard methods. DNA is extracted from the cells, amplified using flanking primers to amplify site 1, site 2 and the region between, and then the amplicons are sequenced. The sequencing in some experiments may be high-throughput sequencing. Any sequence reads with clear mutations in site 1 or site 2 indicate correct processing activity by the flanking tRNAs.

Method 3.3 - Recoding a target gene for the purposes of building a gene drive

(1) Studies are performed that identify suitable target sites with few off-targets within the coding sequence of the gene. The coding sequence of the gene is recoded from the target sites to the end of the coding sequence by changing at least every fourth codon if possible, removing all introns in between, and replacing its 3'UTR with one from another gene with similar expression conditions.

(2) Either guide RNAs (as in Method 3.1) or an RNA-guided DNA nuclease or both are encoded downstream of the 3'UTR as needed for the particular application, but there must be no homology between 3'UTR and any such inserted elements.

Example 4

Provided are methods of building and using serially dependent 1 -dimensional daisy chains of gene drive elements (daisy drive) organisms wherein the terminal element that exhibits drive results in population suppression through either sex-biasing (via targeting a sex chromosome in the germline after the soma-germline division has been specified but before meiosis such that surviving gametes will produce individuals mostly of one sex) or genetic load, (via disrupting genes essential for viability or fertility in one or both sexes in the germline after the soma-germline division has been specified but before meiosis).

Methods of suppressing populations using endonuclease gene drive elements to bias the sex ratio or impose a genetic load have been previously described (Burt, A. 2003 Proc. Roy. Soc. Lond. B. 270,921-8; and Esvelt et al 2014 eLife:e03401, the content of each of which is incorporated herein by reference in its entirety). However, such gene drive elements are inherently self-sustaining and consequently pose risks to all populations of the target species anywhere in the world. New methods are provided herein that are used to limit population suppression to local rather than global populations by creating "daisy chain" gene drive elements that cause local population suppression.

Specifically, methods are provided for the design and construction of a daisy drive chain of any length in which each element requires the prior link in order to drive, and the first element in the chain does not exhibit drive. By including a effector element that imposes genetic load (Fig. 14) or generates a sex -biasing effect, the daisy drive element suppresses the population in the area of release, but because it is a local daisy drive rather than a self- sustaining gene drive, that effect will be limited to the area of release. Any potential configuration of daisy drive elements can be adjusted using methods provided herein to induce a population suppression effect. For example, a daisy chain gene drive can be designed and constructed in which an effector element can replace and therefore eliminate a recessive gene that is important for viability or fertility as would a self- sustaining/global genetic load drive, or a daisy chain gene drive may be designed and constructed that includes multiple guide RNAs that target and disrupt such a gene.

Alternatively, an effector element could be a standard daisy drive element (as described in Example 3, Method 3.0) that encodes both guide RNAs targeting such loci for disruption as well as guide RNAs causing itself to drive. Alternatively, it could include an extra copy of the single gene or set of genes that ensure the organism will develop a one particular sex in the relevant specie; for example, a single copy of the Sry gene in mice causes maleness. Alternatively, the A element could include guide RNAs inducing the RNA-guided DNA nuclease to cut and eliminate a sex chromosome, thereby ensuring that nearly all offspring of A element organisms are of one sex, or that cut and disrupt a recessive gene for viability or fertility. These and other strategies for population suppression can be utilized in methods to design and construct daisy chain gene drives.

Method 4.0 - Suppressing a population via genetic load

(1) Recessive genes are identified that correspond to, in order of preference, sex-specific infertility, infertility, sex-specific viability, or viability by combing the literature or standard genetic techniques.

(2) In the wild-type background, a strain is designed and constructed in which a large portion or all of such a gene is replaced by guide RNAs targeting the wild-type version, such that the guide RNAs exhibit drive in the presence of the RNA-guided DNA nuclease.

(3) Alternatively, a new daisy drive element (per Example 3, Method 3.3) is designed and constructed via genetic recoding, but one that expresses both guide RNAs that will allow it to exhibit drive by targeting the wild-type version of its own locus in the presence of the RNA- guided DNA nuclease and also guide RNAs leading to disruption of one or more genes identified in Step 1. It is possible to target several such genes in the same organism for increased evolutionary robustness.

(4) The resulting strain is crossed with a daisy drive strain created via Example 3, Method 3.0 to create a complete daisy drive strain. Homozygose and employ methods of inhibiting suppression activity in the production facility (Example 3, Method 3.3) as needed. If the suppression method is sex-specific, it is best to use a daisy drive strain in which the proximal element is located within a locus exclusive to the unaffected sex. For example, in Aedes aegypti a daisy drive system disrupting female fertility genes should have the proximal element in the daisy drive chain located within the M locus exclusive to males, thereby ensuring that males carrying the complete drive system are unaffected by suppression, save through mating with sterile females.

(5) Alternatively, add guide RNAs targeting genes identified in Step 1 to element A of the daisy drive strain created via Example 3, Method 3.0. This element is now both element A and an effector.

(6) Information is obtained to determine how many organisms must be released to suppress a target population of a given size to the desired level. The information in some instances may be obtained using cage studies and field trials.

(7) The target population is sampled and the number of organisms required for release is estimated. Based on the estimate, a suitable number of daisy drive organism are released into the target environment to suppress or eliminate the target species from the local area.

Method 4.1 - Suppressing a population by causing drive -carrying organisms to develop as a single sex

(1) Identify a gene whose presence or disruption causes individuals to develop as a particular sex through standard genetic methods (e.g. Sry causes maleness m Mus musculus and Nix in Aedes aegypti, while the loss of fem-3 causes femaleness in C. elegans).

(2) A transgenic organism is created with a new daisy drive element (per Example 3, Method 3.3) via genetic recoding, but one that expresses both guide RNAs that will allow it to exhibit drive by targeting the wild-type version of its own locus in the presence of the RNA-guided DNA nuclease and also guide RNAs leading to disruption of one or more genes identified above. It is possible to target several such genes in the same organism for increased evolutionary robustness.

(3) Alternatively, a transgenic organism is created with a new daisy drive element (per Example 3, Method 3.3) via genetic recoding, but one that expresses guide RNAs that will allow it to exhibit drive by targeting the wild-type version of its own locus in the presence of the RNA-guided DNA nuclease and also encodes one or more genes identified above in Step 1 causing development as a single sex. It is possible to create several such elements in the same organism for increased evolutionary robustness. Methods of inhibiting sex -biasing activity in the organism production facility are employed (Example 3, Method 3.3 or using a tet-OFF system to control expression of genes causing development as one particular sex) as needed.

(4) The resulting strain is crossed with a daisy drive strain created via Example 3, Method 3.0 to create a complete daisy drive strain. Homozygose as needed.

(5) Alternatively, guide RNAs targeting genes identified in Step 1 are added to element A of the daisy drive strain created via Example 3, Method 3.0, or genes causing development as a single sex are added to element A. This element is now both element A and an effector.

(6) A determination is made as to how many organisms must be released to suppress a target population of a given size to the desired level using cage studies and field trials.

(7) The target population is sampled to estimate the number of organisms required for release based on the determination. A suitable number of daisy drive organism are released into the target environment to suppress or eliminate the target species from the local area.

Method 4.2 - Suppressing a population via chromosomal shredding

(1) A set of sequences are identified on either side of the centromere of the sex chromosome that corresponds to the sex a population will be biased against (e.g. the X to male-bias in mice). An off-target-finding software (e.g. sgRNACas9 or GT-Scan) is used as normal to ensure the sites are sufficiently unique in the genome. Ideally, the identified target sequences are unique to the target chromosome but are repeated several times.

(2) In an (N-element) daisy drive (e.g. C-B-A) background, a new target gene (not yet used in the daisy drive) is identified for the daisy drive effector element following Example 2, Method 2.0.

(3) The gene is recoded following procedure of Example 3, Method 3.3 and one or preferably at least two guide RNAs are encoded that target the wild-type version of the target gene and that function with the RNA-guided DNA nuclease encoded in the A element of the daisy drive strain.

(4) An orthogonal RNA-guided DNA nuclease is also encoded such that it is expressed exclusively during late meiosis (see Windbichler N. et al 2011 Nature 473, 212-215; and Port, F. et al., 2014, PNAS vol. 111 no. 29; E2967-E2976, each of which is incorporated by reference herein in its entirety). Guide RNAs for this nuclease are also encoded that target the sequences identified in step 1. The progeny ratio should be biased towards the desired sex; adjust expression conditions until this occurs. It may be possible to use the same nuclease for both functions.

(5) If the proximal element of the daisy drive chain is not encoded within the sex- determining locus or chromosome favored by the A element, the daisy drive strain is simply crossed to wild-type organisms of the non-favored sex to maintain the population and produce organisms for release. See Example 6, Method 6.0 for additional details.

(6) If the proximal element of the daisy drive chain is not encoded within the sex- determining locus or chromosome favored by the A element, a strain is generated that contains only the proximal element of the daisy drive system (e.g. element D for a D-C-B-A system). The daisy drive organisms are crossed to this strain (sorted for the non-favored sex) to maintain the population and produce organisms for release.

(7) A determination is made of the number of gene drive organisms that must be released to suppress a target population of a given size to the desired level using cage studies and field trials.

(8) The target population is sampled to estimate the number of organisms required for release based. A suitable number of daisy drive organism are released into the target environment to suppress or eliminate the target species from the local area. Example 5

Methods of building and using serially dependent 1 -dimensional daisy chains of gene drive elements (daisy drive) organisms with an arbitrary number of elements such that the terminal element targets and recodes a gene important for organismal fitness as it spreads in order to enable the subsequent alteration or suppression of exclusively the previously altered local population at a later date.

Altering a population with a daisy drive permits subsequent precision targeting of the population harboring the introduced sequence with a self-sustaining CRISPR gene drive system that cannot spread beyond the target population. This is a "precision drive" strategy. Method 5.0 - two-stage suppression using guide RNAs only.

1. Recessive genes are identified that corresponding to, in order of preference, sex- specific infertility, infertility, sex-specific viability, or viability by combing the literature or standard genetic techniques.

2. In a wild-type background, one or more of the target genes is replaced with guide RNAs targeting sites within the replaced sequence. Guide RNAs are encoded using expression conditions determined using Example 3, Method 3.1.

3. Laboratory cage studies and/or field trials are performed to determine how many organisms of this strain must be released to suppress or eliminate a population of organisms that already encode an RNA-guided DNA nuclease. 4. It is determined how many organisms of an already-available daisy drive system, such as one created via Example 3, Method 3.0 that has an RNA-guided DNA nuclease as its terminal element, must be released to drive the nuclease gene to fixation in the population to be suppressed or eliminated.

5. Daisy drive organisms are released in suitable numbers to accomplish local fixation. The population into which the daisy drive organisms were released is sampled to ensure the daisy drive has spread to the desired extent.

6. The strain created in step 2 is released in sufficient numbers to suppress or eliminate the population as desired.

7. Alternatively, the population is suppressed by biasing it towards one sex. A suitable target gene or genes are identified using Example 2, Method 2.0. In a wild-type background, the gene(s) are recoded using Example 3, Method 3.3. Just downstream of the new 3'UTR of the gene(s), guide RNAs are encoded that correspond to target sites within the wild-type version of the gene so that it can drive itself in the presence of the appropriate RNA-guided DNA nuclease. A gene is included that ensures carrier organisms develop as a particular sex, or encode guide RNAs that disrupt a gene causing the same outcome, or target sites are identified for chromosomal shredding as in Example 4, Method 4.2, step 1 and an orthogonal RNA-guided DNA nuclease is encoded such that it is expressed exclusively during late meiosis (see Windbichler et al Nature 2011, Port et al PNAS 2014) as well as guide RNAs for this nuclease that target the sequences causing chromosomal shredding. Refer to Example 4, Methods 4.0, 4.1, and 4.2 for additional details on suppression mechanisms.

Method 5.1 - two-stage suppression using genetic load and self-sustaining precision drive

(1) Recessive genes are identified that correspond to, in order of preference, sex-specific infertility, infertility, sex-specific viability, or viability by combing the literature or standard genetic techniques.

(2) One or more of these genes is recoded via Example 3, Method 3.3, ensuring that the recoded sequence contains multiple suitable target sites for a subsequent gene drive system with few or no off-targets in the genome.

(3) Just downstream of the 3'UTR of the gene or genes, guide RNAs are encoded that correspond to target sites within the wild-type version of the gene such that the element drives itself in the presence of an RNA-guided DNA nuclease.

(4) The resulting strain(s) are crossed with a daisy drive strain created via Example 3, Method 3.0 to create a complete daisy drive strain that recodes the target gene(s). Homozygose.

(5) In a wild-type background, one or more of the target genes is replaced with an RNA- guided DNA nuclease encoded using expression conditions determined in Example 2, Method 2.0, and also guide RNAs targeting sites within the first recoded version of the gene, which are encoded using expression conditions determined using Example 3, Method 3.1.

(6) The target population is sampled to estimate the number of organisms. A suitable number of daisy drive organisms created in Step 4 are released in the target environment to recode the nearby population.

(7) Organisms are sampled and sequenced (or checked for a marker gene inserted into the A element of the daisy drive) to verify that a suitable fraction of the relevant population has been recoded. In most cases this entails fixation in the target local population.

(8) Organisms carrying the suppression drive(s) generated in step 5 are released into the target environment. The drive(s) spreads through and suppresses the population recoded with the daisy drive, but not wild-type organisms.

Method 5.2 - two-stage suppression using sex-biasing or sex chromosomal shredding

(1) A suitable target gene or genes is identified using Example 2, Method 2.0.

(2) In a wild-type background, the gene(s) are recoded via Example 3, Method 3.3, ensuring that the recoded sequence contains multiple suitable target sites for a subsequent gene drive system with few or no off-targets in the genome.

(3) Just downstream of the new 3'UTR of the gene(s), guide RNAs corresponding to target sites within the wild-type version of the gene are encoded such that the element drives itself in the presence of an RNA-guided DNA nuclease.

(4) The resulting strain(s) are crossed with a daisy drive strain created via Method 3.0 to create a complete daisy drive strain that recodes the target gene(s). Homozygose.

(5) A suitable target gene or genes is identified using Example 2, Method 2.0. In a wild- type background, the gene(s) are recoded using Example 3, Method 3.3. Just downstream of the new 3'UTR of the gene(s), guide RNAs corresponding to target sites within the wild-type version of the gene are encoded so that it can drive itself in the presence of the appropriate RNA-guided DNA nuclease. A gene is included that ensures carrier organisms develop as a particular sex, or guide RNAs are encoded that disrupt a gene causing the same outcome, or target sites are identified for chromosomal shredding as in Example 4, Method 4.2, step 1 and an orthogonal RNA-guided DNA nuclease is encoded such that it is expressed exclusively during late meiosis (see Windbichler et al Nature 2011, Port et al PNAS 2014) as well as guide RNAs for this nuclease that target sequences causing chromosomal shredding.

(6) The target population is sampled and the number of organisms is estimated. A suitable number of daisy drive organisms created in Step 4 are released in the target environment to recode the nearby population.

(7) Sample organisms are sampled and sequenced (or check for a marker gene inserted into the A or effector element of the daisy drive) and it is verified that a suitable fraction of the relevant population has been recoded. In most cases this entails fixation in the target local population.

(8) Organisms carrying the suppression drive(s) generated in step 5 are released into the target environment. The drive(s) spread through and suppress the population recoded with the daisy drive, but not wild-type organisms.

Example 6

Methods of achieving stable population suppression by locating the first element in the daisy drive chain in a position unique to one sex and suppressing fertility or viability of the other sex.

Daisy drive systems used directly for population suppression will experience a fitness cost limiting their potency. It is possible to ensure that the incidence of the daisy drive remains nearly proportional to the current population by reducing the fertility or viability of one sex while locating the first element of the daisy chain adjacent to a gene unique to the other sex.

For example, a simple C- B- A daisy drive might encode the guide RNAs of the C element adjacent to a male-determining gene (e.g. the Nix gene within the M factor of the dengue vector Aedes aegypti) or a sex chromosome unique to males (e.g. the Y chromosome in the malaria vector Anopheles gambiae). The RNA-guided DNA nuclease is encoded at a B element as is standard for a daisy drive. The A element would include guide RNAs that target and either disrupt or replace female fertility or viability genes. Alternatively, guide RNAs disrupting these genes might be encoded on the B element leaving the A element without guide RNAs of its own.

As a result, daisy drive males would inactivate the female fertility genes during gametogenesis. Their sons would always inherit the C element (as well as B and A thanks to drive) and would suffer minimal fitness penalty, allowing them to repeat the cycle as it occurred in their fathers. Daughters would inherit one copy of the B element and the A element. During gametogenesis, the A element would drive thanks to the presence of the B element, so all offspring of these daughters would inherit a broken copy. If the other parent is a daisy drive male, their daughters will be sterile, thereby suppressing the population.

Method 6.0 - building a daisy drive system for population suppression with reduced fitness cost

(1) Steps 1-5 of Example 3, Method 3.0 are followed to generate a basic daisy drive. Wild-type sequences within the proximal element (e.g. element C if it is a C-B-A drive system) are noted.

(2) A genetic element is identified that is specific to the sex that will NOT be targeted by the drive system (e.g. if the drive system disrupts female fertility, an element specific to males).

(3) In the daisy drive strain, guide RNAs that target the wild-type version of the currently proximal element in the daisy drive chain are encoded within or adjacent to the sex-specific genetic element. Guide RNAs are encoded according to Example 3, Method 3.1.

Example 7

Methods of achieving stable population suppression by using a daisy intermediate to inactivate female fertility genes in a dominant manner.

A variation on the above Examples involves ensuring that the A element exhibits drive in the zygote, thereby ensuring that any female inheriting a single copy of the B element is sterile (or nonviable). This is achieved by arranging for the RNA-guided DNA nuclease encoded in A or optionally a separate nuclease encoded in an effector to be expressed in the zygote and/or the early stages of development. This will cause it to disrupt the wild-type allele of the target gene inherited from the other parent, resulting in sterile or nonviable females. Because the fitness cost to males will be minimal, the introduction of males of this type will cause immediate population suppression proportional to the fraction of daisy drive males; however, it will not increase in the population. This approach may be necessary because there are few genes whose loss causes dominant sterility in a sex.

Daisy chain drive systems designed in this manner permit controlled and persistent population suppression by linking a sex-specific effect to a genetic locus unique to the other sex. For example, female fertility genes such as those recently identified in malarial mosquitoes {Hammond et al 2015 Nat. Biotech. } are targeted by a genetic load daisy drive whose basal element is located on the Y chromosome or an equivalent male-specific locus (Fig. 14). These males suffer no fitness cost due to suppression relative competing wild-type males. The 3-element daisy chain drive system in which female fertility gene disruption occurs early in development creates a male-linked dominant sterile-daughter effect that is otherwise difficult to generate genetically because expressing proteins such as a nuclease from a sex-specific locus or chromosome can be challenging in many organisms, whereas expressing guide RNAs from a polymerase III promoter is possible.

Method 7.0 - building a sex-linked drive system causing dominant sterility in opposite-sex offspring

(1) Steps 1-3 of Example 3, Method 3.0 are followed to generate a strain with just the A element of a daisy drive system, except that the RNA-guided DNA nuclease must be encoded such that active nuclease will be present in the zygote and early embryo (e.g. employ a constitutive or housekeeping promoter such as the actin promoter).

(2) A genetic element is identified that is specific to the sex that will NOT be targeted by the drive system (e.g. if the drive system disrupts female fertility, an element specific to males).

(3) In the strain with element A, guide RNAs are encoded that target the wild-type version of element A within or adjacent to the sex-specific genetic element. Guide RNAs are encoded according to Example 3, Method 3.1. This is element B, which will cause element A to drive.

(4) Steps 1-3 of Example 4, Method 4.0 are followed to generate an effector element, being sure to use genes corresponding to sex-specific infertility.

(5) The resulting strain is crossed to the (B-A) daisy drive strain to create a sex-specific daisy drive strain whose opposite-sex offspring are infertile due to loss of both copies of the target gene(s). Same-sex offspring are (B-A-effector) zygotic daisy drive organisms of nearly normal fitness.

(6) If the above method results in a drive with low homing efficiency and consequent loss of offspring due to non -homologous end-joining in the A target gene, the A or effector element is constructed such that it encodes its own orthogonal RNA-guided DNA nuclease expressed in the germline just after the soma-germline division per Example 3, Method 3.0, as well as guide RNAs directing it to cut the wild-type target gene causing sex-specific infertility.

Example 8 Methods of designing and constructing daisy drive elements in which guide RNAs are embedded within introns of target genes.

Some genes may not be amenable to recoding at the 3' end, or to having their 3'UTR replaced. An alternative method has been developed in which the guide RNAs are encoded within the gene itself. This is most effective when the gene is highly transcribed; fortunately, most haploinsufficient genes chosen as daisy drive targets are ribosomal and are consequently some of the most highly expressed in the cell. However, guide RNAs must be produced from these transcripts without disrupting the function of the gene. A solution has been developed that includes embedding the guide RNAs within introns, separated by tRNAs for efficient processing. The tRNA-processing method has been shown to enable high nuclease activity in fruit flies when driven by strong polymerase II promoters (//dx.doi. org/10.1101/046417); ribozyme-based processing (not suitable for daisy drive due to repetitiveness) works efficiently from within introns (://dx. doi. org/10.1016/j .molcel.2014.04.022). Alternatively, a CRISPR system such as Cpfl that does not require external processing factors may be used. To ensure that the guide RNAs are copied efficiently, the target wild-type gene must be cleaved on both sides of the intron.

Method 8.0 - Embedding daisy drive elements within introns of target genes

(1) For daisy drive elements that would otherwise encode guide RNAs encoded downstream of recoded and 3'UTR-swapped highly-expressed target genes, an alternative design is used that includes inserting a string of alternating tRNAs and guide RNAs into an intron such that tRNAs are on either flank of the string, or alternatively to use a CRISPR nuclease system that does not require external processing factors. Example 3, Method 3.2 is used to identify tRNAs suitable for processing in the relevant organism. It may be necessary to attempt insertion at several such sites in case insertion at one site disrupts splicing; this is less of a risk for larger introns which should be preferred targets. Disrupted splicing is manifested as inviability or extremely poor growth because the target gene should be haploinsufficient.

(2) Nuclease target sites are recoded in the exons on either side of the intron. To reduce the risk of creating a drive-resistant allele through multiple short homology-directed repair events, at least two target sites are included on either side. Optionally, the sequence is recoded between the sites closest to the intron and the boundaries of the intron itself, while leaving the 6-12 bp closest to the splice junction unaffected to minimize the risk of disrupting splicing. (3) The guide RNAs in the upstream element of the daisy chain should target the recoded sites.

Example 9

Methods for building evolutionarily unstable yet robust drive systems through redundancy.

Homing-based gene drive systems are vulnerable to drive-resistant alleles that block drive copying and thus prevent the spread of the drive system. These alleles are generated naturally whenever the endonuclease cut is repaired by non-homologous end-joining, which can create indels or point mutations at the target site that block subsequent cutting. This is why evolutionarily stable drives must target multiple sites within genes important for fitness. However, it is possible to affect large numbers of organisms even without evolutionary stability. A typical rate of HEJ repair is 5% (Gantz, V. & Bier, E. 2015 Science 24

Apr: Vol. 348, Issue 6233, pp. 442-444; Gantz, V. et al., 2015 PNAS Vol. 112 no. 49 E6736- E6743; and Hammond, A. et al., Nat Biotechnol. 2015 Dec 7; doi: 10.1038/nbt.3439, the contents of each of which is incorporated herein by reference in its entirety). Thus, at minimum 5% of the population will be unaffected by the drive system; the share will decline as natural selection favors the resistant alleles over the drive. This precludes suppression drive strategies, but may be acceptable for certain alteration-based requirements. One method of compensating is to build multiple evolutionarily unstable drive systems, each of which targets a single site. There is no need to target a sequence important for fitness as there is no need to select against drive-resistant alleles.

Similar logic has now been applied to daisy drive systems. Because a daisy drive system is not intended to spread indefinitely, each element will only be copied a fixed number of times. This limits the potential for drive-resistant alleles to emerge that block spread. However, this is counterbalanced by the increased number of elements that must be copied, which increases vulnerability to any one drive-resistant allele. Building multiple versions of the daisy drive can compensate, wherein each version can drive the downstream element of every other version. For example, the alpha, beta, and gamma versions of element C encode different guide RNAs, but each is capable of driving either the alpha, beta, or gamma versions of element B. Since resistance to each guide RNA is generated

independently, each version can still cut and replace sequences resistant to the others.

Operationally, each daisy drive element (save optionally for the "A" element encoding the nuclease as well as the effectors) replaces a wild-type "neutral sequence" with multiple target sites for guide RNAs, each which is targeted by a different version. Method 9.0 building redundant evolutionarily unstable drive systems that target the same locus

(1) Instead of building a single homing-based drive system based on an RNA-guided DNA nuclease with multiple guide RNAs, multiple drive systems are built, each of which targets a different sequence or sequences within the same locus. Multiple target sites within a given locus (at least two, preferably more) are identified.

(2) A drive system is constructed by encoding an RNA-guided DNA nuclease with appropriate expression conditions for comparatively efficient homologous recombination as opposed to NHEJ, such as is determined by Example 2, Method 2.0. However, any expression conditions acting upon cells that will eventually compose the germline will do. Additionally, a single highly active promoter is encoded (e.g. identified using Example 2, Method 2.3) that drives a guide RNA targeting one of the target sites. This inserted DNA replaces all target sites identified within the locus.

(3) Additional drive systems are constructed that target all of the other target sites within the locus. Drive systems will not be able to cut and replace one another and so will coexist within the target cell. A drive-resistant allele for one system will not resist another system. Only the gradual accumulation of drive-resistant alleles at all sites will fully block the spread of the drive system. This will eventually occur if the population to be altered is sufficiently large, but it may not matter for many applications.

(4) Organisms comprising all drive systems together are released.

(5) To build equivalent daisy drive systems, this strategy is repeated for each daisy drive element. That is, build multiple daisy drive systems, each of which has one guide RNA per element that targets a different site within the wild-type locus replaced by the next daisy element in the chain (or in the case of effector element(s) that encode their own guide RNA(s), their own locus). It is most effective for daisy elements that replace wild-type "neutral sequences".

Example 10

Family tree analysis was performed and results indicated the power of including additional elements to prepare a daisy chain gene drive. Results are shown in Figs. 4&5. A simple deterministic discrete-generation model of allele frequencies in an isolated panmictic population was prepared and used to analyze the likely effects of seeding at an arbitrary frequency. Each element was assigned a dominant multiplicative fitness cost to account for imperfect homing, gene expression, any off -target cutting, and other losses. The model assumed that each active element was designed to prevent the evolution of drive resistance alleles as previously described (Esvelt et al. 2014 eLife e03401, the contents of which is incorporated herein by reference in its entirety).

Results of the modelling indicated that a C- B- A daisy drives will spread A to near- fixation when released at low but not very low frequencies (Fig. 11 A-B). However, the drives were highly sensitive to the fitness costs incurred by elements B and C (Fig. 11C). Fig. 11 A shows that a daisy drive with 2% fitness cost per upstream element and 10% fitness cost for the final element, seeded at 1%, never approaches fixation. Fig. 1 IB shows that the same drive seeded at 5% would rapidly fix in a non-deterministic model. Fig. 11C shows that if the upstream elements cost 10% each, more organisms would need to be released.

It was determined that adding elements to the daisy chain should help compensate for higher fitness costs, and a formal model was constructed and used to evaluate the

consequences of adding additional elements. It was observed that longer daisy chains lead to much stronger local drive (Fig. 12). At a cost of 5% per daisy drive element, which is readily accessible to current drive systems, four- and five-element daisy drive systems driving a cargo with 10% cost could be released at frequencies as low as 2.5% and 1% respectively and still exceed 99% frequency in less than 20 generations without global spread. Fig 12 illustrates the finding that the A element attains higher frequencies as daisy-chain length increases across a range of fitness costs per upstream element, assuming the final element has a fitness cost of 10%. Fig. 12A shows that with population seeding at 5%, three element chains are sufficient for the A element to reach 99% frequency if the upstream elements have a low fitness cost (2%, left). As the cost increases to 5% (middle), four elements are required, and 10% cost precludes spread above roughly 80%. Fig. 12B shows results that indicated that daisy drives with more elements require fewer organisms to be released in order for the A element to reach a frequency of 99%. Each homing event was assumed to occur with 95% efficiency.

It was determined that for some applications, a periodic release of daisy chain gene drive organisms could provide more cost-effective population control than a single release of the daisy chain gene drive organisms. The model was adjusted to include additional releases in every subsequent generation and analysis of the modelling results indicated that daisy drives can readily alter local populations if repeatedly released in very small numbers (Fig. 13). It was determined that this method of daisy chain drive implementation could be used in applications that must affect large geographic regions over extended periods of time, as for local organism population eradication campaigns. Fig. 13A-B provides graphs illustrating that releasing new organisms in each generation enables faster spread and requires fewer organisms per release. The numerical simulations depicted in Fig. 13A-B are identical to Fig. 11, except the initial release is repeated each generation. The final construct is assumed to have a 10% fitness cost. Fig. 13 A shows that three- four- or five-element daisy drives can spread constructs with upstream elements having fitness costs of 2% (left) or 5% (middle) to 99% frequency. Four- or five-element drives are sufficient when the upstream elements have higher (10%) fitness costs. Fig. 13B indicates that repeated release at very low frequency (0.1%)) is sufficient for spread of the final element to 99% frequency for upstream elements having fitness costs of 2% (left) or 5% (middle), while >1% repeated release is required for higher cost (10%) elements.

Additional modeling examined generic daisy drive systems in which all non-"A" daisy elements comprised sequences encoding guide RNAs that replaced wild-type neutral sequences, wherein each such daisy element caused both the next element and the "A" element to exhibit drive (Fig. 9). This is the drive system created using Method 3.0.2. The model showed that while drive-resistant alleles are generated, they do not substantially interfere with the spread of the "A" element (which does not tolerate drive -resistant alleles due to inclusion of a recoded essential gene). The inclusion of additional daisy drive elements (e.g. G-F-E-D-C-B-A) was always beneficial to overall spread as long as each element added only a small fitness cost (Fig. lOA-C).

Example 11

It was determined that any recombination event that moved one or more guide RNAs within an upstream element of the chain into any downstream element would convert a linear daisy drive chain into a self-sustaining CRISPR gene drive 'necklace' (Fig. 5). Methods to reduce the likelihood of such recombination event were developed and tested.

It was determined that a way to reliably prevent such recombination events was to eliminate regions of homology between the elements. Means to remove promoter homology were developed that included use of different U6, HI, or tRNA promoters for each element {see Port et al. (2014) PNAS, Ranganathan et al (2014) Nat. Comm.

doi : 10.1038/ncomms5516, Mefferd et al (2015) RNA doi : 10.1261 /rna.051631.115 } , by expressing multiple guide RNAs from a single promoter using tRNA processing {see Xie et al. (2015) PNAS doi: 10.1073/pnas.1420294112 , Port and Bullock (2016) bioRxiv

doi: 10.1101/046417} or by connecting a pair of sgRNAs by a short linker. However, it was recognized that each element still required guide RNAs that were over 80 base pairs in length, which precluded safe and stable daisy drive designs.

Methods

Guide RNA Design: S. pyogenes Cas9

Examination was made of existing data on guide RNA variants and corresponding activities as well as the crystal structure of S. pyogenes Cas9 in complex with sgRNA to identify bases that would likely tolerate mutation. Using this information, a set of 20 sgRNAs were constructed and assays for activity (see below) using only two replicates to identify sequence changes that were harmful to activity. These experiments indicated that the large insertion found in sgRNAs from closely related bacteria was well-tolerated in only one case. The insertion was removed and additional sgRNAs designed. All of the designed sgRNA candidates were constructed and assayed to identify those with sufficiently high activity. For experiments requiring additional highly divergent sgRNAs, such as daisy suppression drives in which the "A" element encodes many guide RNAs that disrupt multiple recessive fertility genes at multiple sites, a more comprehensive approach to activity profiling is performed that examines additional candidate guide RNAs. Fig. 6 shows the sequences of candidate guide RNAs that were designed, constructed, and tested for activity (SEQ ID NOs: 3-35).

Measuring Guide RNA Activity: S. pyogenes Cas9

HEK293T cells were grown in Dulbecco's Modified Eagle Medium (Life

Technologies) fortified with 10% FBS (Life Technologies) and Penicillin/Streptomycin (Life Technologies). Cells were incubated at a constant temperature of 37°C with 5% C0 ₂ . In preparation for transfection, cells were split into 24-well plates, divided into approximately 50,000 cells per well. Cells were transfected using 2μ1 of Lipofectamine 2000 (Life

Technologies) with 200ng of dCas9 activator plasmid, 25ng of guide RNA plasmid, 60ng of reporter plasmid and 25ng of EBFP2 expressing plasmid.

Fluorescent transcriptional activation reporter assays were performed using a modified version of addgene plasmid #47320, a reporter expressing a tdTomato fluorescent protein adapted to contain an additional gRNA binding site lOObp upstream of the original site. gRNAs were co-transfected with reporter, dCas9-VPR, a tripartite transcriptional activator fused to the C-terminus of nuclease-null Streptococcus pyogenes Cas9, and an EBFP2 expressing control plasmid into HEK293T cells. 48 hours post-transfection, cells were analyzed by flow cytometry. In order to exclusively analyze transfected cells, cells with less than 10 ³ EBFP2 expression were ignored. The preliminary screen of the initial 20 designs was performed with only two replicates to identify critical bases. Experiments evaluating the final set of sgRNA sequences were performed with six biological replicates.

Guide RNA Design: Acidaminococcus Cpfl

Examination was made of existing data on the crRNAs of various Cpfl relatives to identify bases that would likely tolerate mutations. Using this information, variants with single mutations in the four bases preceding the stem or within the loop were constructed. Similarly, mutants that changed both paired bases at a position in the stem were constructed. In some variants, these mutations were combined. Variant repeats were inserted into arrays of ten crRNAs, each paired with a spacer sequence with a matching protospacer in a unique target plasmid. Repeat variants were located in different positions and paired with different spacers to control for position and spacer effects.

Measuring Guide RNA activity: Acidaminococcus Cpfl

Plasmid exclusion assays were performed by transforming cells expressing AsCpfl and an array or control cells lacking an array with the target plasmids, plating, and measuring the difference in the number of colonies. The effectiveness of each variant was recorded for different positions and spacers consistently active, defined as exhibiting >10-fold exclusion in all cases, identified.

Results

To identify highly active guide RNA sequences with minimal homology to one another, known tracrRNA, crRNA, and alternative sgRNA sequences for CRISPR systems related to that of S. pyogenes were compared and variable regions were identified. Dozens of sgRNA variants that had been designed to be as divergent from one another as possible were created. These candidate sgRNAs were assayed using a sensitive tdTomato-based transcriptional activation reporter identified 15 different sgRNAs with activities comparable to the standard version (Figs. 6&13). This set of minimally homologous sgRNAs should enable stable daisy drive systems of up to 5 elements with 4 sgRNAs per driving element. Future studies will need to examine the stability of the resulting daisy drive in an animal model. These divergent guide RNAs will also enable global CRISPR gene drive elements to overcome the problem of 'drive-resistant alleles' that cannot be cut and replaced. Targeting multiple adjacent sequences within genes important for fitness was previously described as a solution for this problem {Esvelt et al (2014) eLife}, but repetitive elements even within a single drive construct often prove unstable {Simoni et al (2014) Nucl. Acids Res.}.

Example 12

Guide RNA activity measurement using a transcriptional activation reporter assay

The activity of candidate guide RNAs was measured and determined using a transcriptional activation reporter using dCas9-VPR.

(1) Mammalian cells were grown using standard conditions (e.g. HEK293T cells were grown in Dulbecco's Modified Eagle Medium (Life Technologies) fortified with 10% FBS (Life Technologies) and Penicillin/Streptomycin (Life Technologies), incubated at a constant temperature of 37°C with 5% C0 ₂ ).

(2) The cells were split into 24-well plates, divided into approximately 50,000 cells per well and then transfected with plasmids encoding:

(a) dCas9-VPR (or the equivalent dead-nuclease transcriptional activator variant of the RNA-guided DNA-binding protein nuclease matching the guide RNAs to be tested),

(b) the guide RNA to be evaluated,

(c) a reporter plasmid comprising a minimal promoter and one or more protospacer binding site upstream of a gene encoding a fluorescent protein, and

(d) a control plasmid expressing a different fluorescent marker gene as a transfection control marker.

(3) The transfections were carried out as follows: using 2μ1 of Lipofectamine 2000 (Life Technologies) with 200ng of dCas9 activator plasmid, 25ng of guide RNA plasmid, 60ng of reporter plasmid and 25ng of EBFP2 expressing plasmid. The reporter plasmid was a modified version of addgene plasmid #47320, a reporter expressing a tdTomato fluorescent protein adapted to contain an additional gRNA binding site lOObp upstream of the original site, the activator is da tripartite transcriptional activator fused to the C-terminus of nuclease- null Streptococcus pyogenes Cas9).

(4) After transfection, the cells were analyzed using flow cytometry to measure activity, and any cells that didn't fluoresce due to the presence of the transfection control marker were ignored. (5) Optionally, if a library of guide RNAs is assayed at the same time, use fluorescent- assisted cell sorting (FACS) is used to sort for plasmids encoding highly active guide RNAs which are then sequenced to identify. Guide RNA activity measurement using plasmid exclusion

The activity of candidate guide RNAs was measured and determined using a plasmid exclusion assay.

1) E. coli cells expressing AsCpfl and either a guide RNA array or an empty vector were separately grown and rendered competent using standard methods.

2) Target plasmids carrying protospacers corresponding to each spacer in the array or no sequence were constructed and sequence-confirmed.

3) Target plasmids were individually transformed into the competent cells by heat shock, recovered for 2 minutes on ice and then 1 hour at 37°C. Dilutions were plated on LB agar plates containing antibiotics selecting for all three plasmids and grown for 24 hours at 37C. 4) The number of colony-forming units for each construct was measured for each plasmid and type of competent cells. Equivalent numbers of the control plasmid lacking a protospacer indicated that both types of cells were equally competent. The ratio of colony- forming units between the two types of cells was used as a metric of Cpfl plasmid exclusion activity.

5) Exclusion indices of >10-fold (e.g. there were consistently >10x as many colonies in the absence of the array) were recorded as active.

6) Variants that were consistently active regardless of spacer and array position were identified.

7) Optionally, a more comprehensive library-based approach can be adopted using the plasmid exclusion assay to exclude plasmids encoding an inducible toxin which kills transformed cells grown in the presence of inducer. Alternatively, variant crRNAs from the library can be paired with a spacer conferring resistance to a lytic bacteriophage, enabling active crRNAs to be isolated by exposing the bacteria to the targeted bacteriophage. Example 13

Studies were performed and a daisy drive system was constructed. In the study, the daisy drive system was prepared in C. elegans.

Materials and Methods: Daisy drive system preparation in C. elegans

1) A large number (over 100) of adult C. elegans were injected with all three daisy drive vectors (see Fig. 17), Pcfj601 (available directly from Addgene as Plasmid #34874 ) for Mosl transposase, and pre-complexed cas9 protein from the Alt-R CRISPR-Cas9 System of IDT (Integrated DNA Technologies) to aid with integration. (See user guide on IDT website: //www.idtdna.com/pages/products/genome-editing/crispr-genome -editing/crispr-cas9- genome-editing) See Table 2 for guide RNAs used.

Table 2. Guide RNAs used with cas9 protein to aid with integration of daisy drive cassettes

The daisy drive vectors used are shown in Figure 17 and were as follows:

Daisy link 'Α' : which contained myo3-mCherry-unc54 UTR flanked by 500 bp of both 5' and 3' homology sites for Cku80.

Daisy link B: which contained Pmyo2-GFP-unc54UTR and guides targeting Cku80. It is flanked by both 5' and 3' homology arms to fog2.

EM-Hera: Daisy link C: which contained Prpll28 + BFP + let-858 UTR + gRNA targeting fog-2. Daisy link C was the bottom link of the daisy chain and was integrated randomly into the genome via Mosl transposase. Daisy link C was not driven by any gRNA whatsoever. 2) Worms were isolated that expressed all three colors of fluorescence (RGB) in the Fl generation.

3) Glowing Fl worms were injected a second time with vectors of the missing color to maximize probability of integration.

4) Twenty (20) RGB worms were isolated in or before the young adult stage. This step occurred before any of the 20 had laid any eggs.

5) RGB worms from step '3' were divided into two groups of ten (10) worms each. One group of 10 was a control group and the worms in that group were left unchanged. The other group of 10 worms, the "Daisy" group, received injections of additional cas9 protein (ΙΟμΜ) upon reaching adulthood. The injections were performed for both gonads of the worms.

It was expected that the worms in the parental generation (P0) would be heterozygous for all three gene drive elements. The injection of the cas9 protein was done to initiate "drive" activity.

6) Following step 5, the worms were left for three (3) days to lay eggs and for the Fl generation of the worms to mature.

7) The prepared C. elegans daisy drive organisms were assessed for genomic copy number and daisy drive activity. The assessment included qPCR analysis as described below.

Assessment for genomic copy number and daisy drive activity

C. elegans are known to retain injected genetic material in extrachromosomal arrays for a number of generations post initial injection. Therefore simple counting of fluorescence in the Fl generation of the prepared C. elegans was not sufficient to determine drive activity. To assess drive activity in the prepared C. elegans, qPCR was used to determine the number of integrated copies of each gene. For the qPCR studies, primer pairs were designed that amplified across the junction between the inserting gene drive cassette and the existing genomic DNA. This ensured that only integrated gene drive cassettes were accounted for in the assessment. Plasmid vectors containing the target template for qPCR were diluted to an appropriate concentration of 2.42 and 4.84 zeptomoles in lx TE buffer and used as positive controls. Negative controls were created by substituting distilled water as amplification template.

The appropriate concentration of DNA used for positive controls are matched to the theoretical concentration of single worm DNA extractions according to the following reasoning. An adult C. elegans contains 959 (diploid) somatic cells and 1000-2000 germ cells (haploid). On this basis, it was assumed that a homozygote worm contained 2918 copies of the gene drive cassette and that a heterozygote worm contained 1459 copies. This translates to 4.84 and 2.42 zeptomoles respectively. 168 worms were picked from each of the Fl generation progeny from the two experiments, the 'daisy' and 'control' groups, described above. Each worm was suspended individually in lOuL of lysis buffer following the Williams et al. protocol (Williams BD, et. al., (1992) Genetics Jul; 131(3):609-24, and //github.com/mfitzp/theolb/blob/master/molecular-biology/c-e legans-single-worm-pcr.rst). The worms were then flash-frozen in an ethanol / dry ice slurry. The frozen worms were heated to 65° C and then heated to 95° C. The resulting prepared single worm DNA extract was stored at 4° C until used the qPCR procedure. qPCR procedure

qPCR is performed on an bio-Rad qPCR cfx384 instrument with the intensity threshold for Cq set at 0.2. The same program is used for all qPCR experiments: 95°C for 3 minutes followed by 40 cycles of (95°C for 10 seconds and 55 °C for 1 minute). qPCRs are performed using the KAPA Sybr Fast qPCR kit following manufacturer's instructions. For the 384-well plate format we used, each qPCR reaction was made up of 5μΙ. of Kapa Master Mix, 0.2 μΙ_, of each 10 μΜ primer, 2.6 μΙ_, of distilled water, and 2 μΙ_, of genomic DNA extract from each of the single worms. For positive and negative controls, the worm genomic DNA extract was replaced with either plasmid vectors diluted to the appropriate

concentration or water as described below.

For positive controls, 2.42 and 4.84 zeptomoles of positive control vectors were added to row 'A' of a 384 well plate to represent homo and heterozygosity. The remaining wells were filled with single worm DNA extract from the Fl generation. Thus, each 96-well plate represented a random sampling of progeny from two individual parents. It was expected that a ~1 cycle difference in time to 0.2 fluorescence intensity on qPCR between heterozygotes and homozygotes of the daisy drive cassettes. It was expected that most, if not all of the fluorescing worms of the "control" group to be heterozygotes. It was expected that most, if not all, of the fluorescing worms of the "daisy" group to be homozygotes.

The Daisy Element Ά', or the ultimate link of the prepared daisy chain, was expected to exhibit the behavior described above only if the daisy drive system was working as designed.

Table 3. Mean 'Cq' values of the data groups. Note: 'n' value for Control 'A' is lower than 168 because a number of samples failed to run and were excluded from the mean calculation. Data points from both "daisy" and "control" groups that overlap with negative controls were removed from data table.

Control 'B' 14.32553 0.288308 144

Positive control 'B' 13.4535 0.16743 48

Daisy ^* C ^* 24.43035 0.463744 136

Control ^* C ^* 24.40731 0.484118 156

Positive control 'C 23.81716 4.157946 48

Results

Results of the qPCR are shown in Figs. 18 and 19 and in Table 3, and demonstrated that the daisy drive system as described was working as designed. The data showed that almost all of the fluorescing "control" group worms were heterozygotes and almost all of the fluorescing worms of the "daisy" group were homozygotes.

Statement for all Examples

Means for designing constructing, integrating, and implementing such systems of the invention as well as preparing organism strains and releasing organisms of such strains, etc. that include such systems of the invention is carried out using the teaching presented herein, and in certain instances in conjunction with methods, components, and/or elements known in the art. Equivalents

Although several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present invention is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto; the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, and/or methods, if such features, systems, articles, materials, and/or methods are not mutually inconsistent, is included within the scope of the present invention.

All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

The indefinite articles "a" and "an," as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

The phrase "and/or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified, unless clearly indicated to the contrary.

All references, patents and patent applications and publications that are cited or referred to in this application are incorporated herein in their entirety herein by reference. What is claimed is: