Patent Description:
Alternative methods have been developed to accelerate the process of genome modification by directly injecting DNA or mRNA of site-specific nucleases into the one cell embryo to generate DNA double strand break (DSB) at a specified locus in various species. DSBs induced by these site-specific nucleases can then be repaired by either error-prone non-homologous end joining (NHEJ) resulting in mutant mice and rats carrying deletions or insertions at the cut site. If a donor plasmid with homology to the ends flanking the DSB is coinjected, high-fidelity homologous recombination can produce animals with targeted integrations. Because these methods require the complex designs of zinc finger nucleases (ZNFs) or Transcription activator-like effector nucleases (TALENs) for each target gene and because the efficiency of targeting may vary substantially, no multiplexed gene targeting has been reported to date.

Thus, improved methods for producing genetically modified cells to generate animals, such as pigs, are needed for potential sources of organs for transplantation.

The invention is defined by the scope of the appended claims.

Described herein is the use of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated (Cas) proteins (CRISPR/Cas) system to achieve highly efficient and simultaneous targeting of multiple nucleic acid sequences in cells.

Aspects of the present disclosure are directed to the modification of genomic DNA, such as multiplex modification of DNA, in a porcine cell (e.g., stem cell, somatic cell, germ line cell, zygote) using one or more guide RNAs (ribonucleic acids) to direct a Cas9 enzyme having nuclease activity expressed by the cell, to a target porcine endogenous retrovirus (PERV) pol gene on the DNA (deoxyribonucleic acid) wherein the Cas9 enzyme cuts the DNA and an exogenous donor nucleic acid is inserted into the DNA, such as by homologous recombination. Aspects of the present disclosure include cycling or repeating steps of DNA modification in a porcine cell to create a porcine cell having multiple modifications of DNA within the cell. Modifications can include insertion of exogenous donor nucleic acids. Modifications can include deletion of endogenous nucleic acids.

Multiple nucleic acid sequences can be modulated (e.g., inactivated) by a single step of introducing into a porcine cell, which expresses a Cas9 enzyme, nucleic acids encoding a plurality of gRNAs, such as by co-transformation, wherein the gRNAs are expressed and wherein each gRNA in the plurality guides the Cas9 enzyme to a particular site of the DNA, the Cas9 enzyme cuts the DNA. According to this aspect, many alterations or modification of the DNA in the cell are created in a single cycle.

The porcine cell expressing the Cas9 enzyme has been genetically altered to express the enzyme by introducing into the cell a nucleic acid encoding the Cas9 enzyme and which can be expressed by the cell. In this manner, aspects of the present disclosure include cycling the steps of introducing gRNA into a porcine cell which expresses the Cas9 enzyme, introducing exogenous donor nucleic acid into the cell, expressing the RNA, forming a co-localization complex of the gRNA, the Cas9 enzyme and the DNA, and enzymatic cutting of the DNA by the Cas9 enzyme. Insertion of a donor nucleic acid into the DNA is also provided herein. Cycling or repeating of the above steps results in multiplexed genetic modification of a porcine cell at multiple loci, i.e., a porcine cell having multiple genetic modifications.

DNA binding proteins or enzymes within the scope of the present disclosure include a protein that forms a complex with the guide RNA and with the guide RNA guiding the complex to a double stranded DNA sequence wherein the complex binds to the DNA sequence. The enzyme is a Cas9 protein.

This aspect of the present disclosure may be referred to as co-localization of the RNA and DNA binding protein to or with the double stranded DNA. In this manner, a DNA binding protein-guide RNA complex may be used to cut multiple sites of the double stranded DNA so as to create a porcine cell with multiple genetic modifications, such as disruption of all copies of a gene.

According to certain aspects, a method of making multiple alterations to target PERV pol genes in a porcine cell expressing a Cas9 enzyme that forms a co-localization complex with RNA complementary to the target DNA and that cleaves the target DNA in a site specific manner is provided including (a) introducing into the porcine cell a nucleic acid encoding a guide ribonucleic acid (gRNA) complementary to all or a portion of a target sequence within a PERV pol gene in the porcine cell and which guide the Cas9 enzyme to the target sequences within the PERV pol genes, wherein the gRNA and the Cas9 enzyme are members of a co-localization complex for the target DNA, wherein the gRNA and the Cas9 enzyme co-localize to the target DNA, the Cas9 enzyme cleaves the target DNA to produce altered DNA in the cell, and repeating step (a) multiple times to produce multiple alterations to the DNA in the cell.

According to the invention there is provided a method of disrupting target porcine endogenous retrovirus (PERV) pol genes in a porcine cell with a targeting efficiency of at least <NUM>% comprising: introducing into the porcine cell a guide ribonucleic acid (gRNA) complementary to all or a portion of a target sequence within a PERV pol gene in the porcine cell, and a nucleic acid sequence that encodes a Cas9 protein; and maintaining the porcine cell under conditions in which the Cas9 protein is continually expressed; wherein the Cas9 protein and gRNA form a complex and the complex binds to the complementary target sequences within the PERV pol genes and disrupt the target PERV pol genes in the porcine cell.

In the methods described herein, the introducing step can comprise transfecting the cell with the gRNA sequence and the nucleic acid sequence that encodes the Cas9 protein.

In some embodiments, the gRNA sequences, the nucleic acid sequence that encodes the Cas9 protein, or a combination thereof are introduced into a genome of the cell.

In some embodiments, the expression of the Cas9 protein is induced.

In the methods described herein, the porcine cell is from an embryo. The porcine cell can be a stem cell, zygote, or a germ line cell. In embodiments where the porcine cell is a stem cell, the stem cell is an embryonic stem cell or pluripotent stem cell. In other embodiments, the porcine cell is a somatic cell.

The target nucleic acid sequences comprise porcine endogenous retrovirus (PERV) pol genes.

The methods described herein disrupt target PERV pol genes with a targeting efficiency of at least <NUM>%. In some embodiments, all copies of the pol gene in the cell are inactivated.

In some embodiments, the gRNA sequence can be about <NUM> to about <NUM> nucleotides. For example, the gRNA sequence can be about <NUM> to about <NUM> nucleotides.

In another aspect, an engineered porcine cell can comprise a plurality of PERV pol genes; and one or more exogenous nucleic acid sequences that comprise a portion that is complementary to all or a portion of one or more target nucleic acid sequences of the plurality of PERV pol genes; wherein each of the plurality of PERV pol genes of the cell are modulated.

In some embodiments, all or substantially all copies of the pol gene in the cell are inactivated.

According to one aspect, the gRNA is between about <NUM> to about <NUM> nucleotides. According to one aspect, the gRNA is between about <NUM> to about <NUM> nucleotides.

According to one aspect, the gRNA is a tracrRNA-crRNA fusion.

According to one aspect, the DNA is genomic DNA, mitochondrial DNA, viral DNA, or exogenous DNA.

Aspects of the present invention are directed to the use of CRISPR/Cas9, for nucleic acid engineering. Described herein is the development of an efficient technology for the generation of animals (e.g., pigs) carrying multiple mutated genes. Specifically, the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated genes (Cas genes), referred to herein as the CRISPR/Cas system, has been adapted as an efficient gene targeting technology e.g., for multiplexed genome editing. Demonstrated herein is that CRISPR/Cas mediated gene editing allows the simultaneous inactivation of <NUM> copies of the porcine endogenous retrovirus (PERV) pol gene in a porcine kidney epithelial cell line (e.g., PK15) with high efficiency. Co-injection or transfection of Cas9 mRNA and guide RNA (gRNA) targeting PERVs into cells generated a greater than <NUM> fold reduction in PERV transmission to human cells with biallelic mutations in both genes with an efficiency of up to <NUM>%. Shown herein is that the CRISPR/Cas system allows the one step generation of cells carrying inactivation of all copies of PERV. In certain embodiments a method described herein generates cell and pigs, with inactivation of target PERV pol genes with a targeting efficiency of at least <NUM>%, <NUM>%, <NUM>%, or more.

The shortage of organs for transplantation is a major barrier to the treatment of organ failure. While porcine organs are considered promising, their use has been checked by concerns about transmission of porcine endogenous retroviruses (PERVs) to humans. Here, the eradication of all PERVs in a porcine kidney epithelial cell line (PK15) was performed. It was first determined the PK15 PERV copy number to be <NUM>. Using CRISPR-Cas9, all <NUM> copies of the PERV pol gene were disrupted and demonstrated a > <NUM>-fold reduction in PERV transmission to human cells using our engineered cells. This study showed that CRISPR-Cas9 multiplexability can be as high as <NUM> and demonstrates the possibility that PERVs can be inactivated for clinical application to porcine-to-human xenotransplantation.

Pig genomes contain from a few to several dozen copies of PERV elements. Unlike other zoonotic pathogens, PERVs cannot be eliminated by biosecure breeding. Prior strategies for reducing the risk of PERV transmission to humans have included small interfering RNAs (RNAi), vaccines, and PERV elimination using zinc finger nucleases and TAL effector nucleases, but these have had limited success. Here, the successful use of the CRISPR-Cas9 RNA-guided nuclease system can be used to inactivate all copies of the PERV pol gene and effect a <NUM>-fold reduction of PERV infectivity of human cells.

To design Cas9 guide RNAs (gRNAs) that specifically target PERVs, the sequences of publically available PERVs and other endogenous retroviruses in pigs (Methods) were analyzed. A distinct clade of PERV elements (<FIG>) were identified and determined there to be <NUM> copies of PERVs in PK15 cells using droplet digital PCR (<FIG>). Two Cas9 guide RNAs (gRNAs) were designed that targeted the highly conserved catalytic center of the pol gene on PERVs (<FIG>, Fig. S1). The pol gene product functions as a reverse transcriptase (RT) and is thus essential for viral replication and infection. It was determined that these gRNAs targeted all PERVs but no other endogenous retrovirus or other sequences in the pig genome (Methods).

Initial experiments showed inefficient PERV editing when Cas9 and the gRNAs were transiently transfected (Fig. S2). Thus a PiggyBac transposon system was used to deliver a doxycycline-inducible Cas9 and the two gRNAs into the genome of PK15 cells (Fig. S2-<NUM>). Continuous induction of Cas9 led to increased targeting frequency of the PERVs (Fig. S5), with a maximum targeting frequency of <NUM>% (~<NUM> PERV copies per genome) observed on day <NUM> (Fig. S5). Neither higher concentrations of doxycycline or prolonged incubation increased targeting efficiency (Fig. S4,<NUM>), possibly due to the toxicity of non-specific DNA damage by CRISPR-Cas9. Similar trends were observed when Cas9 was delivered using lentiviral constructs (Fig. S6). The cell lines that exhibited maximal PERV targeting efficiencies were genotyped. <NUM> different insertion and deletion (indel) events centered at the two gRNA target sites (<FIG>) was observed. Indel sizes ranged from <NUM> to 148bp; <NUM>% of indels were small deletions (<9bp). The initial deep sequencing results was validated with Sanger Sequencing (Fig. S7).

Single cells from PK15 cells with high PERV targeting efficiency were sorted using flow cytometry and genotyped the pol locus of the resulting clones via deep sequencing. A repeatable bimodal (<FIG>, S8-<NUM>) distribution was observed with ~<NUM>% of the clones exhibiting high levels of PERV disruption (<NUM>%-<NUM>%), and the remaining clones exhibiting low levels of editing (<<NUM>%). Individual indel events were examined in the genomes of these clones (<FIG>, Fig. S10-<NUM>). For the highly edited clones (Clone <NUM>, <NUM>%; Clone <NUM>, <NUM>%; Clone <NUM>, <NUM>%; Clone <NUM>, <NUM>%), only <NUM>-<NUM> unique indel patterns in each clone (<FIG>, S11) were observed. In addition, there was a much higher degree of repetition of indels within each clone than across the clones (Fig. S25), suggesting a mechanism of gene conversion in which previously mutated PERV copies were used as templates to repair wild-type PERVs cleaved by Cas9 (<FIG>, Fig. S25). Mathematical modeling of DNA repair during PERV elimination (Fig. S26) and analysis of expression data (Fig. S22-<NUM>) supported this hypothesis and suggested that highly edited clones were derived from cells in which Cas9 and the gRNAs were highly expressed.

Next, unexpected genomic rearrangements had occurred as a result of the multiplexed genome editing was examined. Karyotyping of individual modified clones (Fig. S12-S14) indicated that there were no observable genomic rearrangements. <NUM> independent genomic loci with at most 2bp mismatches to each of the intended gRNA targets were examined and observed no non-specific mutations (Fig. S27). This suggests that our multiplexed Cas9-based genome engineering strategy did not cause catastrophic genomic instability.

Last, disruption of all copies of PERV pol in the pig genome could eliminate in vitro transmission of PERVs from pig to human cells was examined. No detection of RT activity in the cell culture supernatant of the highly modified PK15 clones (Fig. S15) was observed, suggesting that modified cells only produced minimal amounts of PERV particles. Co-culture of WT and highly modified PK15 cells with HEK <NUM> cells were tested directly for transmission of PERV DNA to human cells. After co-culturing PK15 WT and HEK <NUM> cells for <NUM> days and <NUM> days (Fig. S16-<NUM>), PERV pol, gag, and env sequences in the HEK <NUM> cells were detected (<FIG>). The estimated frequency of PERV infection was approximately <NUM> PERVs/<NUM> human cells (<FIG>). However, PK15 clones with > <NUM>% PERV pol targeting exhibited up to <NUM>-fold reduction of PERV infection, similar to background levels (<FIG>). These results were validated with PCR amplification of serial dilutions of HEK293 cells that had a history of contact with PK15 clones (<FIG>, S18-<NUM>). PERVs in single HEK293 cells isolated from the population co-cultured with minimally modified Clone <NUM> was consistently detected, but could not distinctly detect PERVs in <NUM> human cells from the population co-cultured with highly modified Clone <NUM>. Thus, PERV infectivity of the engineered PK15 cells had been reduced by up to <NUM> fold.

In summary, it was successfully targeted the <NUM> copies of PERV pol in PK15 cells and demonstrated greatly reduced in vitro transmission of PERVs to human cells. While in vivo PERV transmission to humans has not been demonstrated, PERVs are still considered risky and our strategy could completely eliminate this. As no porcine embryonic stem cells exist, this system will need to be recapitulated in primary porcine cells and cloned into animals using somatic cell nuclear transfer. Moreover, simultaneous Cas9 targeting of <NUM> loci in single pig cells without salient genomic rearrangement was achieved. To our knowledge, the maximum number of genomic sites previously reported to be simultaneously edited has been six. Our methods thus open the possibility of editing other repetitive regions of biological significance.

PERV copy number quantification: Droplet Digital PCR ™ PCR (ddPCR™) was usd to quantify the copy number of PERVs according to the manufacturer's instructions (Bio-Rad). Briefly, genomic DNA (DNeasy Blood & Tissue Kit, Qiagen) from cultured cells was purified, digested <NUM> ng genomic DNA with MseI (10U) at <NUM> for <NUM> hour, and prepared the ddPCR reaction with <NUM>µl 2X ddPCR Master mix, <NUM>µl of <NUM> target primers & <NUM> target probe (VIC), 1µl of <NUM> reference primers & <NUM> reference probe (FAM), 5ng digested DNA, and water to total volume of <NUM>µl. The sequence of the primers and the probe information can be found in Extended Data Table <NUM>.

CRISPR-Cas9 gRNAs design: MUSCLE was used to carry out a multiple sequence alignment of <NUM> endogenous retrovirus found in the porcine genome. A phylogenetic tree of the sequences was built and identified a clade that included the PERVs (see <FIG>). The R library DECIPHER was used to design specific gRNAs that target all PERVs but no other endogenous retroviral sequences.

Cell culture: PK15 were maintained in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) high glucose supplemented with <NUM>% fetal bovine serum (Invitrogen), and <NUM>% penicillin/streptomycin (Pen/Strep, Invitrogen). All cells were maintained in a humidified incubator at <NUM> and <NUM>% CO<NUM>.

PiggyBac-Cas9/2gRNAs construction and cell line establishment: PiggyBac-Cas9/2gRNAs construct is derived from a plasmid previously reported in Wang et al (<NUM>). Briefly, a DNA fragment encoding U6-gRNA1-U6-gRNA2 was synthesized (Genewiz) and incorporated it into a PiggBac-Cas9 construct. To establish PK15 cell lines with PiggyBac-Cas9/2gRNAs integration, <NUM>•<NUM><NUM> PK15 cells was transfected with <NUM>µg PiggyBac-Cas9/2gRNAs plasmid and <NUM>µg Super PiggyBac Transposase plasmid (System Biosciences) using Lipofectamine <NUM> (Invitrogen). To enrich for the cells carrying the integrated construct, <NUM>µg/mL puromycin was added to the transfected cells. Based on the negative control, puromycin was applied to wild type PK15 cells, it was determined that the selection completed in <NUM> days. The PK15-PiggyBac cell lines were maintained with <NUM>µg/mL puromycin hereafter. <NUM>µg/ml doxycycline was applied to induce Cas9 expression.

Lentivirus-Cas9/2gRNAs construction and cell line establishment: Lenti-Cas9/2gRNAs constructs were derived from a plasmid previously reported (<NUM>). A DNA fragment encoding U6-gRNA1-U6-gRNA2 was synthesized (Genewiz) and incorporated it into a Lenti-Cas9-V2. To generate lentivirus carrying Lenti-Cas9/2gRNAs, ~ <NUM>•<NUM><NUM> 293FT HEK cells was transfected with <NUM>µg Lenti-Cas9-gRNAs and <NUM>µg ViraPower Lentiviral Packaging Mix (Invitrogen) using Lipofectamine <NUM>. The lentiviral particles were collected <NUM> hours after transfection, and the viral titer was measured using Lenti-X GoStix (Takara Clonetech). ~ <NUM><NUM> lentiviral particles to ~ <NUM>•<NUM><NUM> PK15 cells were transduced and conducted selection by puromycin to enrich transduced cells <NUM> days after transduction. The PK15-Lenti cell lines were maintained with <NUM>µg/mL puromycin thereafter.

Genotyping of colonized and single PK15 cells: PK15 cultures were dissociated using TrypLE (Invitrogen) and resuspended in PK15 medium with the viability dye ToPro-<NUM> (Invitrogen) at a concentration of <NUM>-<NUM>•<NUM><NUM> cells/ml. Live PK15 cells were single-cell sorted using a BD FACSAria II SORP UV (BD Biosciences) with <NUM> nozzle under sterile conditions. SSC-H versus SSC-W and FSC-H versus FSC-W doublet discrimination gates and a stringent '<NUM>/<NUM>/<NUM> single-cell' sorting mask were used to ensure that one and only one cell was sorted per well. Cells were sorted in <NUM>-well plates with each well containing 100µl PK15 medium. After sorting, plates were centrifuged at <NUM> for <NUM>. Colony formation was seen <NUM> days after sorting and genotyping experiment was performed <NUM> weeks after FACS.

To genotype single PK15 cells without clonal expansion, the PERV locus was directly amplified from sorted single cells according to a previously reported single cell genotyping protocol (<NUM>). Briefly, prior to sorting, all plastics and non-biologic buffers were treated with UV radiation for <NUM>. Single cells were sorted into <NUM>-well PCR plates with each well carrying <NUM>. 5µl 10X KAPA express extract buffer (KAPA Biosystems), <NUM>µl of 1U/µl KAPA Express Extract Enzyme and <NUM>µl water. The lysis reaction was incubated at <NUM> for <NUM> and inactivated the reaction at <NUM> for <NUM>. All reactions were then added to 25µl PCR reactions containing <NUM>. 5µl 2X KAPA <NUM> fast (KAPA Biosystems), <NUM> PERV illumina primers (Methods Table2), and <NUM>. Reactions were incubated at <NUM> for <NUM> followed by <NUM> cycles of <NUM>, <NUM>; <NUM>, <NUM> and <NUM>, <NUM>. To add the Illumina sequence adaptors, 5µl of reaction products were then added to <NUM>µl of PCR mix containing <NUM> of <NUM> KAPA HIFI Hotstart Readymix (KAPA Biosystems), <NUM> primers carrying Illumina sequence adaptors and 7µl water. Reactions were incubated at <NUM> for <NUM> followed by <NUM>-<NUM> cycles of <NUM>, <NUM>; <NUM>, <NUM> and <NUM>, <NUM>. PCR products were checked on EX <NUM>% gels (Invitrogen), followed by the recovery of <NUM>-400bp products from the gel. These products were then mixed at roughly the same amount, purified (QIAquick Gel Extraction Kit), and sequenced with MiSeq Personal Sequencer (Illumina). Deep sequencing data was analyzed and determined the PERV editing efficiency using CRISPR-GA (<NUM>).

Targeting efficiency estimation: a custom pipeline was built to estimate the efficiency of PERV inactivation. Briefly, the pol gene was amplified and sequenced via Illumina Next Generation Sequencing using PE250 or PE300. First, the two overlapping reads were combined using PEAR (<NUM>) and mapped to the reference region using BLAT. After mapping, the reads were grouped into sets containing specific combinations of haplotypes (see Extended Data <FIG>), and indel types. Read sets with representation lower than <NUM>% of the total number of mapped reads were discarded. Finally, the mapping output was parsed to call the different insertions and deletions as described in Güell et al (<NUM>).

RNA-seq analysis: The susScr3 pig genome and Ensembl transcripts were obtained from the UCSC Genome Brower Database. RNA-Seq reads were mapped to the reference genome using the STAR software (<NUM>) and the RPKM of the transcripts were quantified using BEDTools (<NUM>). Differential expression analysis was performed in R using the DESeq2 package (<NUM>), and gene set enrichment analysis was carried out by the GSEA software (<NUM>), with gene set definitions obtained from the software's website.

Reverse transcriptase (RT) assay: To test the RT activity of the PK15 cells and modified PK15 clones (<NUM> highly and <NUM> lowly modified clones), <NUM>•<NUM><NUM> cells were plated in T75 cm<NUM> flasks, and collected the supernatant <NUM> days after seeding. The media was filtered using a <NUM> Millex-HV Syringe Filter (EMD Millipore Corporation), and the filtered supernatant was concentrated at <NUM> for <NUM> using Amicon Ultra-<NUM> Centrifugal Filter Unit (EMD Millipore Corporation). The concentrated supernatant was ultra-centrifuged at <NUM>,<NUM> rpm for <NUM>. The supernatant was carefully removed, and the virus pellet was collected and lysed with <NUM>µl of <NUM>% NP40 at <NUM> for <NUM>.

The RT reaction was conducted using the Omniscript RT Kit (Qiagen). The total volume of the reaction was <NUM>µl, which contained <NUM> RT buffer, <NUM> dNTPs, <NUM> Influenza reverse primer (<NUM>' CTGCATGACCAGGGTTTATG <NUM>') (SEQ ID NO: <NUM>), <NUM> units of RnaseOUT (Life Technology, Invitrogen), <NUM> units of SuperRnase Inhibitor (Life Technologies), <NUM>µl of sample lysis and <NUM> ng of IDT-synthesized Influenza RNA template which was rnase resistant in both <NUM>' and <NUM>' end. The RNA template sequence was <NUM>' rA*rA*rC*rA*rU*rGrGrArArCrCrUrUrUrGrGrCrCrCrUrGrUrUrCrArUrUrUrUrArGrArArAr UrCrArArGrUrCrArArGrArUrArCrGrCrArGrArArGrArGrUrArGrArCrArUrArArArCrCrCrUr GrGrUrCrArUrGrCrArGrArCrCrU*rC*rA*rG*rU*rG <NUM>' (* phosphodiester bond) (SEQ ID NO: <NUM>). After the RT reaction was completed, the RT product was examined by PCR using Influenza forward (<NUM>' ACCTTTGGCCCTGTTCATTT <NUM>') (SEQ ID NO: <NUM>) and Influenza reverse primers (sequence shown as above). The expected size of the amplicon was 72bp.

HEK293-GFP cell line establishment: The Lenti-GFP construct was derived from the plasmid pLVX-IRES-ZsGreen1 (Clontech. Catalog No. <NUM>; PT4064-<NUM>). To generate the lentivirus carrying Lenti-GFP, ~ <NUM>•<NUM><NUM> 293FT HEK cells were transfected with 3µg of pVX-ZsGreen plasmid and <NUM>µg of ViraPower Lentiviral Packaging Mix (Invitrogen) using Lipofectamine <NUM> (Invitrogen). Lentiviral particles were collected <NUM> hours after transfection, and the viral titer was measured using Lenti-X GoStix (Takara Clonetech). ~ <NUM><NUM> lentivirus particles to ~ <NUM>•<NUM><NUM> HEK293 cells were transfected and conducted selection by puromycin to enrich the transduced cells <NUM> days after transduction. The <NUM>-GFP-Lenti cell lines were maintained with <NUM>µg/mL puromycin thereafter.

Infectivity test of PK15 WT to HEK293-GFP: <NUM>•<NUM><NUM> cells of Lenti-GFP-293FT HEK cells and <NUM>•<NUM><NUM> PK15 WT cells were cultured together in a <NUM>-well plate. In parallel, <NUM>•<NUM><NUM> PK15 WT cells were cultured alone in another well as a control. The puromycin selection experiment was done by adding <NUM>µg/ml of the antibiotic for <NUM> days. The time point was determined when no viable cells in the control well and approximately <NUM>% GFP positive cells in the experimental well as the time point when the puromycin selection was completed to purify lenti-GFP-293FT human cells. Cells from the 293FT HEK/PK15 WT co-culture were collected at different time periods. The genomic DNA was extracted using (DNeasy Blood & Tissue Kit, Qiagen) from cultured cells of the <NUM>-GFP WT, PK15 WT and the co-cultured cells. The genomic DNA concentration was measured using a Qubit <NUM> Fluorometer (Invitrogen), and <NUM> ng from each sample was used as DNA template for PCR. In all, <NUM>µL of the genomic DNA were added to <NUM>µL of a PCR mix containing <NUM>µL 2X KAPA Hifi Hotstart Readymix (KAPA Biosystems) and <NUM> of primers as listed in Methods Table <NUM>. Reactions were incubated at <NUM> for <NUM> followed by <NUM> cycles of <NUM>, <NUM>; <NUM>, <NUM> and <NUM>, <NUM>. PCR products were visualized on EX <NUM>% gels (Invitrogen) and observed for bands of <NUM>-<NUM> base pairs.

Quantification of PERV copy numbers infected in HEK293-GFP cells: qPCR was performed to quantify the PERV copy number in HEK293-GFP cells. Genomic DNA of PK15 WT cells of different amounts was used as the template for the qPCR reactions. Reactions were conducted in triplicate using KAPA SYBR FAST qPCR Master Mix Universal (KAPA Biosystems). PERV pol, env, gag primers, human ACTB and pig GGTA1 primers (Methods Table <NUM>) were added to a final concentration of <NUM>. Reactions were incubated at <NUM> for <NUM> (enzyme activation) followed by <NUM> cycles of <NUM>, <NUM> (denaturation); <NUM>, <NUM> (annealing/extension). The logarithm of the genomic DNA amount linearizes with the quantification cycle (Cq). pol, gag, env primers were used to examine for presence of PERVs. Pig GGTA1 primers served to control for potential porcine genome contaminants in human cells after infection. All experiments were conducted in triplicate.

Infectivity Assay of the Modified PK15 clones to HEK293-GFP: <NUM>•<NUM><NUM> cells of HEK293-GFP cells and <NUM>•<NUM><NUM> cells of the high modified (<NUM>, <NUM>, <NUM>, <NUM>) clones and low modified clones (<NUM>, <NUM>) were co-cultured in a <NUM>-well plate for <NUM> days. To isolate the HEK293-GFP cells in order to examine for PERV elements, the GFP positive cells were double sorted to purify the human cell populations.

To quantify the PERV infectivity of different clones to HEK293-GFP cells, both qPCR assays and PCR assays were conducted on series diluted HEK293-GFP cells after sorting. For the qPCR assays, the genomic DNA (DNeasy Blood & Tissue Kit, Qiagen) was extracted from double sorted HEK293-GFP cells. The genomic DNA concentration was measured using Qubit <NUM> fluorometer (Invitrogen). In all, <NUM> ng of the genomic DNA was added to <NUM>µL of KAPA SYBR FAST qPCR reaction (KAPA Biosystems) using PERV pol, env, gag and pig GGTA primers respectively (Extended Data Table <NUM>). The qPCR procedure was performed as described above. For the series dilution assay, purified HEK293-GFP cells were sorted (<NUM> cell/ well, <NUM> cells/well, <NUM> cells/well, <NUM> cells/well) into <NUM>-well PCR plates for direct genomic DNA extraction and PCR reactions. Briefly, cells were sorted into <NUM>µL lysis reaction including <NUM>µL of 10X KAPA Express Extract Buffer, <NUM>µL of <NUM> U/µl KAPA Express Extract Enzyme and <NUM>µL of PCR-grade water (KAPA Biosystems). The reactions were then incubated at <NUM> for <NUM> (lysis), then at <NUM> for <NUM> (enzyme inactivation). Subsequently, the PCR master mix was prepared. In all, <NUM>µL of the genomic DNA lysis was added to <NUM> different <NUM>µL of KAPA Hifi Hotstart PCR reactions (KAPA Biosystems) using <NUM> PERV pol, env, gag primers, and pig GGTA primers, respectively (Extended Data Table <NUM>). The reactions were incubated at <NUM> for <NUM> (initial denaturation) followed by <NUM> cycles of <NUM>, <NUM> (denaturation); <NUM>, <NUM> (annealing), <NUM>, <NUM> sec/kb, then <NUM>, <NUM>/kb (final extension). (KAPA Biosystems). The PCR products were visualized on <NUM> well E-Gel® Agarose Gels, SYBR® Safe DNA Gel (Invitrogen).

CRISPR-Cas9 off-target analysis: whole genome sequencing (WGS) data was obtained for PK15 (untreated cell line) and clone <NUM> (highly edited clone). To investigate potential off-target effects of the Cas9/2gRNAs, the reference sequence (Sus Scrofa <NUM>) was searched for sites that differed from the <NUM> bp sequences targeted by the two gRNAs by only <NUM> or <NUM> bp. <NUM> such sites were identified and extracted them, together with <NUM> bp of their neighboring regions (Fig. S1). BLAT was used to map the WGS reads to the extracted reference sequences and searched for potential indel patterns that had emerged in Clone <NUM> as a result of off-target effects. An average coverage of <NUM>-<NUM> X per loci was obtained. Reads with <<NUM> bp matches with the reference sequence were excluded. In case of reads that mapped to the reference sequence with multiple alignment blocks, which could indicate the presence of indels, reads whose alignment blocks contained <<NUM> bp matches were excluded with the reference sequence. After inspecting the remaining mapped reads, there was no detection any off-target indel patterns present in clone <NUM>. Another challenge was to comprehensive searches for off-targets here is that the Sus Scrofa genome is still neither complete nor completely assembled, limiting the ability to do whole-genome analysis.

Mathematical model of DNA repair process interaction during cumulative PERV inactivation: In this study PERV elements were inactivated by mutations generated by DNA repair processes in response to dsDNA cuts created by Cas9. It is generally understood that dsDNA cuts may be repaired either by non-homologous end joining (NHEJ) or Homologous Repair (HR), and that while HR can create precise copies of a DNA template sequence at the cut site given the presence of a template with suitable homology arms, NHEJ can generate mutations (especially indels) and is often considered "error prone. " However, there is also evidence that NHEJ can also repair dsDNA cuts highly accurately (<NUM>, <NUM>), and the relative rates of mutated vs. perfect repair by NHEJ have never been precisely measured. Especially when efficient targeted nucleases such as Cas9 are expressed for protracted time periods, perfect repair of a cut site by either NHEJ or HR would regenerate a target site that could be cut again. A plausible hypothesis is that the process of perfect repair and re-cutting would occur repeatedly until a mutation arose that destroyed the nuclease's ability to recognize the target site. To explore the way these repair modalities might work together during the course of PERV elimination, their interactions as a Markov process was modeled. Specifically, it was assumed:.

The Markov model computes the probability distribution <MAT>, where <MAT>is the probability that there are i target-ablating mutations at cut c, where c = <NUM>, <NUM>, <NUM>. It is assumed that the initial condition P(<NUM>) = (<NUM>,<NUM>, ···,<NUM> ), i.e., that all targets begin as wild-type. The N+<NUM>-by-N+<NUM> transition matrix M is given as <MAT> <MAT> <MAT>.

Finally, P(c+<NUM>) = p(c)M for c = <NUM>,<NUM>,<NUM>,···.

The formulas for M assume proposition ii above and state in mathematical terms that the number of mutated sites in a cell remains unchanged whenever a cut at a wild-type site is repaired perfectly by NHEJ or by HR using another copy of the wild-type template (formula for M(i, i)), but increases by one if the cut is repaired by mutagenic NHEJ or by HR using a previously mutated site (formula for M(i, i + <NUM>)).

The model incorporates two notable simplifications to actual biology: (i) Target recognition is assumed to be binary - either the nuclease recognizes a target or it does not. This is tantamount to assuming that small mutations that still support target recognition do not substantially alter wild-type cutting rates and therefore can be effectively lumped together with wild-type sites. (ii) HR repairs using mutated vs. wild-type templates are assumed to be equally efficient. Modifications could be made to the model to address these simplifications, but this is not considered here. It is also worth noting that, formally, given assumption ii above, the Markov process should actually stop should the condition <MAT> be reached for some value of c, since at this point no wild-type sites remain to be cut, whereas what happens instead mathematically is that cuts continue but the model remains in a fixed state. Finally, the model effectively represents the mutation count distribution as a function of independent variable c (number of cuts) and not as a function of time. No prediction is made regarding the time rates of DNA repair or PERV site elimination, although time can be assumed to increase monotonically with c.

To analyze PERV elimination through the Markov model, N was always set to <NUM>. However, since the relative efficiencies of perfect vs. mutagenic NHEJ repair are unknown (as noted above), and because relative rates of mutagenic NHEJ vs. HR repair can vary widely depending on cell state and type, the mutation count distributions for a discrete grid covering the complete two-dimensional space of all possible parameter values for n, m, and h, (<NUM> parameter combinations in all) was computed. The model was implemented both as a MatLab (Mathworks, Waltham) script and as an R script using the library markovchain (available as Supplemental Files modelMarkov. m, modelMarkov. R, respectively).

In addition to computing the mutation count distribution via the Markov model for particular parameter values, the MatLab script performed random simulations of the NHEJ and HR repair processes throughout a series of K cuts, allowing bivariate distributions of the numbers of total mutations vs. distinct NHEJ events to be estimated, illustrated in Fig. <NUM> B-C. The R script was used to estimate the most likely state of the system over the grid of n, m, and h combinations described above. K was varied depending on the computation. As illustrated in Fig. S27, the invariable result of the model was a unimodal distribution of mutation counts whose mean advanced towards fixation at N mutations with c, and in Figures S27 B. C, K was set to a value high enough to demonstrate fixation. For the calculation of the most likely state of the system over the n, m, and h grid, K was set to <NUM>, <NUM>, <NUM>, or <NUM>, and <NUM> simulations were conducted for each parameter combination.

Illumina Miseq data with PERVs elements genotyping data has been uploaded to the European Nucleotide Archive (ENA) hosted by the European Bioinformatics Institute (EBI) with the submission reference PRJEB11222.

Claim 1:
A method of disrupting target porcine endogenous retrovirus (PERV) pol genes in a porcine cell with a targeting efficiency of at least <NUM>% comprising:
introducing into the porcine cell a nucleic acid encoding a guide ribonucleic acid (gRNA) complementary to all or a portion of a target sequence within a PERV pol gene in the porcine cell;
introducing into the cell a nucleic acid encoding a Cas9 protein; and
maintaining the porcine cell under conditions in which the Cas9 protein is continuously expressed;
wherein the Cas9 protein and gRNA form a complex and the complex binds to the complementary target sequences within the PERV pol genes and disrupt the target PERV pol genes in the porcine cell.