Comprehensive Physiology Wiley Online Library

A Practical Guide to Genome Editing Using Targeted Nuclease Technologies

Full Article on Wiley Online Library



ABSTRACT

Genome engineering using programmable nucleases is a rapidly evolving technique that enables precise genetic manipulations within complex genomes. Although this technology first surfaced with the creation of meganucleases, zinc finger nucleases, and transcription activator‐like effector nucleases, CRISPR‐Cas9 has been the most widely adopted platform because of its ease of use. This comprehensive review presents a basic overview of genome engineering and discusses the major technological advances in the field. In addition to nucleases, we discuss CRISPR‐derived base editors and epigenetic modifiers. We also delve into practical applications of these tools, including creating custom‐edited cell and animal models as well as performing genetic screens. Finally, we discuss the potential for therapeutic applications and ethical considerations related to employing this technology in humans. © 2019 American Physiological Society. Compr Physiol 9:665‐714, 2019.

Comprehensive Physiology offers downloadable PowerPoint presentations of figures for non-profit, educational use, provided the content is not modified and full credit is given to the author and publication.

Download a PowerPoint presentation of all images


Figure 1. Figure 1. Trends in publication of genome engineering technologies. Data were collected by searching for the terms “gene editing” OR “genome editing” OR “genome engineering” on PubMed for the years 1980 through 2017 and downloading the data file for “Results by year” on the Search results page. The year of the first publication using each nuclease platform for genome editing in mammalian cells is labeled.
Figure 2. Figure 2. Homodimer and monomer meganucleases bound to their DNA target sites. Meganucleases are relatively small, globular proteins composed of structurally integrated DNA binding and cleavage domains. Cleavage is achieved by (A) homodimerization at palindromic or pseudopalindromic sequences, indicated by shading of DNA bases, or (B) monomeric two‐domain proteins, which recognize nonpalindromic sites. These monomeric proteins consist of two domains that function similarly to single‐domain meganucleases, joined into a single meganuclease by an amino acid linker. With either configuration, a DNA DSB occurs at the cleavage site between the two DNA binding domains.
Figure 3. Figure 3. Pair of zinc finger nucleases (ZFNs) bound to their DNA target sites. ZFNs are composed of tandem zinc finger DNA‐binding domains fused to the FokI nuclease domain by an amino acid linker. A pair of four finger ZFNs is depicted here, with two finger modules connected by a short linker. Each zinc finger motif recognizes and binds 3 to 4 base pairs of DNA (grouped by color, ZF1‐ZF4) in a ZFN half site. Two ZFNs must bind in an “antiparallel” manner and with the appropriate spacing in order for the FokI domains to dimerize and cleave the DNA in the spacer region between the two half‐sites. “N” and “C” indicate N‐terminus and C‐terminus of each ZFN.
Figure 4. Figure 4. Pair of transcription activator‐like effector nucleases (TALENs) bound to their DNA target sites. TALENs are comprised of tandem TAL effector DNA binding repeats flanked by an N‐terminal domain (N) and a half repeat followed by a C‐terminal domain (C). This DNA binding domain is fused to the FokI nuclease domain by an amino acid linker. A pair of TALENs each targeting 17 bp is depicted binding a TALEN half site. Each TALEN repeat recognizes and binds a single base pair of DNA (colored bars), and the repeat variable residues (RVD) are shown in the context of the repeat peptide. Two TALENs must bind in an “antiparallel” manner with the appropriate spacing in order for the FokI domains to dimerize and cleave the DNA in the spacer region between the two half‐sites. “N” and “C” indicate N‐terminus and C‐terminus of each TALEN.
Figure 5. Figure 5. CRISPR immune response pathway. The CRISPR‐Cas immune response occurs in three phases: adaptive, biogenesis, and targeting. During the adaptive phase, short PAM containing DNA sequences (protospacers) from invading virus are recognized and integrated into the bacterial genome within the CRISPR array as spacers. The CRISPR array contains several spacers separated by short direct repeat sequences, and is located within the CRISPR locus downstream of several CRISPR associated (Cas) genes. During the biogenesis phase, the spacers are transcribed and processed into mature crRNA. In the targeting phase, crRNAs complex with Cas. This RNA‐Cas complex is guided by the crRNA to specifically bind and cleave a complimentary portion of target DNA, creating a double‐strand break (DSB) that silences the gene and prevents infection.
Figure 6. Figure 6. CRISPR‐Cas9 CRISPR RNA (crRNA) and single‐guide RNA (sgRNA) bound to their DNA target sites. CRISPR‐Cas9 nucleases are comprised of a Cas9 nuclease complexed with a guide RNA. This guide can either be (A) a sequence‐specific crRNA (purple) and a general tracrRNA (red) or (B) a single gRNA (sgRNA) developed by truncation and fusion of crRNA and tracrRNA sequences. The Cas9 protein has two lobes, REC and NUC, and two domains with helicase and nuclease activity: the HNH domain, which interacts with the target strand of DNA, and the RuvC domain, which interacts with the nontarget strand. The DNA target site has 20 nucleotides that are complementary to the first 20 nucleotides of the crRNA/sgRNA. The PAM site (orange) is found on the nontarget genomic DNA strand immediately after the target site.
Figure 7. Figure 7. Comparison of Cas proteins with distinct mechanisms. (A) Cas9 binds to genomic DNA complementary to its guide RNA (crRNA:tracrRNA duplex depicted) and cleaves the target site DNA in between the third and fourth base pair from its 5′‐NGG‐3′ protospacer adjacent motif (PAM), resulting in blunt ends at the double‐strand break. (B) The crRNA that binds to Cas12a contains a stem loop secondary structure at its 5' end. Cas12a binds to genomic DNA complementary to its crRNA and cleaves the complementary strand of the target site in between the 23rd and 24th nucleotide (nt) away from its T‐rich PAM, and between the 18th and 19th nt away from the PAM on the non‐complementary strand, resulting in 5 nt staggered overhangs. (C) Cas13a binds to mRNA complementary to its crRNA and cleaves the mRNA outside of the target site. Cas13a requires a protospacer flanking sequence (PFS) of A, U, or C.
Figure 8. Figure 8. Double‐strand break repair pathways used for genome editing. The two main repair pathways used by the cell to repair double‐strand breaks (DSBs) are nonhomologous end joining (NHEJ) and homology‐directed repair (HDR). NHEJ, the preferred repair pathway, is designed to repair DSBs with precise end joining; however, targeted nuclease activity can introduce direct ligation of an exogenous sequence, or insertions or deletions at the cleavage site, resulting in gene disruption. Simultaneous DSBs introduced at two nearby sites can induce large deletions or inversions, and simultaneous DSBs introduced at two sites on different chromosomes can induce translocations. HDR naturally repairs DSBs using a sister chromatid as a repair template. Delivery of an exogenous donor template with homology flanking the target site, along with a targeted nuclease, can introduce precise nucleotide modifications or sequence insertions via the HDR repair pathway.
Figure 9. Figure 9. On‐target assays for indel quantification. (A) Mismatch detection assays require PCR amplification of the target site from the genomic DNA of a cell pool or clone. These PCR amplicons are denatured at 95°C, then allowed to reanneal. Amplicons with indels that reanneal to wild‐type amplicons or amplicons with different indels will result in DNA bulges that are recognized and cleaved by a mismatch cleavage enzyme. Cleavage products can be visualized and quantified via gel electrophoresis. (B) Computational analysis of Sanger sequencing via TIDE or ICE also begins with PCR amplification of the target site. PCR amplicons are then analyzed by Sanger senquencing. Computational analysis is used to compare the edited pool sequence to the wild‐type sequence and quantify the editing activity. (C) Indel detection by amplicon analysis uses a fluorescent tri‐primer system to fluorescently label the PCR amplicons. Capillary electrophoresis is used to separate the fluorescent amplicons based on size, and the migration time and fluorescent intensity is used to quantify the size and relative concentration of indels. (D) Fluorescent reporter plasmids can be delivered along with targeted nuclease reagents to estimate gRNA activity. One such plasmid contains the sequence for GFP and RFP, with the target site in between such that the RFP sequence is out of frame, expressing the GFP but not the RFP protein. Cleavage at the target site will induce indels in between the two fluorescent proteins, some of which will cause a frameshift allowing expression of RFP. Cleavage and editing can be measured via RFP and GFP fluorescence intensity. (E) Another fluorescent reporter plasmid contains the sequence for a fluorescent protein that is disrupted by the target site flanked by direct repeat sequences (DR). Cleavage at the target site induces repair via the SSA pathway, which restores the fluorescent protein expression. (F) High‐resolution melting analysis DNA dependent fluorescent dye is allowed to bind to target site DNA amplicons. Fluorescence intensity is measured as the amplicons are slowly denatured over time. Amplicons with indels will denature at different rates than the wild‐type amplicons.
Figure 10. Figure 10. On‐Target assays for confirming desired editing events. (A) The restriction fragment polymorphism (RFLP) assay confirms successful knockin by introducing a new restriction enzyme site along with the desired mutation. After PCR amplification of the target site, the DNA is incubated with the restriction enzyme. The restriction enzyme will only cleave amplicons with successful integration and introduction of the restriction site. The cleavage products can be visualized via gel electrophoresis. The wild‐type amplicon and the cleavage products will be present for heterozygous knockins, and the cleavage products alone will be present in clones with homozygous knockin alleles. (B) Junction PCRs can be used to confirm the successful integration of the desired modification. Primers are designed to amplify the junctions between the genomic DNA and the integrated sequence. The presence of an amplicon of the correct size for both junctions confirms a successful knockin event. (C) Western blot or ELISA can be used to confirm the successful knockin of a mutation or sequence integration that is recognizable by an antibody, such as a mutation that causes a structural change or the addition of a protein domain. (D) Where appropriate, a fluorescent tag can be included along with the desired mutation, which can be used to detect successful integration.
Figure 11. Figure 11. On‐target assays for indel quantification and confirming desired editing events (knockin and knockout). (A) Computational analysis of Sanger sequencing via TIDER or ICE begins with PCR amplification of the target site. PCR amplicons are then analyzed by Sanger sequencing. Computational analysis is used to compare the edited pool sequence to the wild‐type sequence and quantify the editing activity. TIDER requires comparison to a positive control sequence for analysis of HDR, while ICE does not. (B) For NHEJ and HDR analysis via targeted genome sequencing, edited genomic DNA is first PCR amplified using site‐specific primers with adaptors, then PCR amplified using indexing primers. These amplicons are sequenced using next‐generation sequencing, and both NHEJ and HDR events can be analyzed using the same data set.
Figure 12. Figure 12. Modifications to the CRISPR‐Cas9 platform to reduce off‐target editing. (A) Modifications made to the gRNA to increase specificity. Tru‐sgRNAs are truncated gRNAs with complementarity lengths of 17 or 18 nucleotides. GG‐sgRNAs have two target site independent, 5’ guanine residues, which have been shown to increase sgRNA specificity. (B) Alternative Cas9 architectures to reduce off‐target cleavage. Nickases contain a point mutation that renders one of the nuclease domains inactive (indicated with gray oval) resulting in the creation of a single‐strand DNA nick. Paired nickases are depicted in the PAM‐out orientation. With proper orientation and gRNA offset, paired nickases create two single‐stranded DNA nicks, which can result in a DSB with staggered overhangs. RNA‐guided FokI nucleases (RFNs) are composed of catalytically dead Cas9 (dCas9) fused to the FokI nuclease domain. The FokI nuclease requires dimerization for cleavage. DSBs are created between RFNs with proper orientation and spacing.
Figure 13. Figure 13. CRISPR epigenetic regulators. (A) dCas9 can repress gene expression by blocking transcription initiation or transcription elongation. Fusion of a Krüppel‐associated box (KRAB) domain to the dCas9 protein improves this repressive activity in mammalian cells. CRISPR activators can generally be categorized into dCas9 protein fusion (B) or gRNA aptamer fusion strategies (C). (B) VP64 (VP16 × 4) and VPR (VP64, p65, and Rta domains in tandem) have been fused directly to dCas9 for use as a gene activator. The SunTag system fuses several GCN4 peptides to the dCas9 protein. GCN4 peptides are recognized by a GCN4 scFv antibody fused to a VP64 domain. (C) Replacing the tetraloop and stemloop of the gRNA with MS2 aptamers allows recruitment of tandem p65‐HSF1 domains fused to MCP, which binds to the MS2 aptamer. This was termed the synergistic activation mediator (SAM) system. The Casilio system appends several PBS domains to the 3’ end of the gRNA. VP64 domains are recruited to the gRNA via fusion to a Pumilio/FBF (PUF) RNA‐binding domain, which binds to the corresponding PUF binding site (PBS). The male‐specific bacteriophage‐2 (MS2) aptamer or the com aptamer have also been appended to the 3’ end of the gRNA. VP64 is recruited to the MS2 domain and KRAB fused to COM is recruited to the com aptamer. This system was used to create orthogonal CRISPR transcriptional regulators for simultaneous gene activation and repression.
Figure 14. Figure 14. Pooled and arrayed screening strategies. (A) A pooled screen is typically conducted using lentiviral particles. The library pool is delivered to cells with and without Cas9 expression. A selective pressure is applied to the cells, and next‐generation sequencing is used to analyze the gRNA sequences that have been enriched (positive selection) or depleted (negative selection). (B) In an arrayed screen, gRNAs can be delivered as RNA, plasmid, or lentivirus. Cas9 expressing cells are plated into 96‐ or 384‐well format, and each gRNA in the library is delivered into an individual well. Wells in which the desired phenotype occurs can then be easily traced back to the gRNA and targeted gene.
Figure 15. Figure 15. Cell line creation workflow. Steps involved in using CRISPR‐Cas9 to develop a targeted genome‐modified cell line. Reagents are designed and validated (1‐5), delivered to cells (6), screened for desired modification(s) (7‐9), and the final genetically modified clone is expanded, genotype confirmed, and cryopreserved (10). The funnel depicts the large number of input cells (orange) with a rare genome edit (green), which is isolated and expanded to form the cell line of interest.
Figure 16. Figure 16. Animal model creation workflow. Steps involved in using CRISPR‐Cas9 to develop a targeted genome‐modified animal model. Reagents are designed and validated (1‐5), injected into fertilized oocytes (6), transferred to a pseudopregnant female (7), and after birth (8), the offspring are screened for the desired modification(s) (9). Selected animals must be bred to confirm germline transmission of the genome edits and to establish the line (10).
Figure 17. Figure 17. Using programmable nucleases for human ex vivo therapy. For human ex vivo therapy using programmable nucleases, target cells are (A) harvested from a patient and (B) expanded in culture. (C) Nuclease(s) and donor DNA, (if required) are delivered to patient cells to allow for the desired genomic modification. Donor DNA can be delivered via plasmid, single stranded DNA, single stranded oligonucleotide donor, or virus. (D) Genetically modified cells are then reintroduced into the patient (autologous transplant) and need to home to the appropriate niche(s) and expand to provide a therapeutic effect.


Figure 1. Trends in publication of genome engineering technologies. Data were collected by searching for the terms “gene editing” OR “genome editing” OR “genome engineering” on PubMed for the years 1980 through 2017 and downloading the data file for “Results by year” on the Search results page. The year of the first publication using each nuclease platform for genome editing in mammalian cells is labeled.


Figure 2. Homodimer and monomer meganucleases bound to their DNA target sites. Meganucleases are relatively small, globular proteins composed of structurally integrated DNA binding and cleavage domains. Cleavage is achieved by (A) homodimerization at palindromic or pseudopalindromic sequences, indicated by shading of DNA bases, or (B) monomeric two‐domain proteins, which recognize nonpalindromic sites. These monomeric proteins consist of two domains that function similarly to single‐domain meganucleases, joined into a single meganuclease by an amino acid linker. With either configuration, a DNA DSB occurs at the cleavage site between the two DNA binding domains.


Figure 3. Pair of zinc finger nucleases (ZFNs) bound to their DNA target sites. ZFNs are composed of tandem zinc finger DNA‐binding domains fused to the FokI nuclease domain by an amino acid linker. A pair of four finger ZFNs is depicted here, with two finger modules connected by a short linker. Each zinc finger motif recognizes and binds 3 to 4 base pairs of DNA (grouped by color, ZF1‐ZF4) in a ZFN half site. Two ZFNs must bind in an “antiparallel” manner and with the appropriate spacing in order for the FokI domains to dimerize and cleave the DNA in the spacer region between the two half‐sites. “N” and “C” indicate N‐terminus and C‐terminus of each ZFN.


Figure 4. Pair of transcription activator‐like effector nucleases (TALENs) bound to their DNA target sites. TALENs are comprised of tandem TAL effector DNA binding repeats flanked by an N‐terminal domain (N) and a half repeat followed by a C‐terminal domain (C). This DNA binding domain is fused to the FokI nuclease domain by an amino acid linker. A pair of TALENs each targeting 17 bp is depicted binding a TALEN half site. Each TALEN repeat recognizes and binds a single base pair of DNA (colored bars), and the repeat variable residues (RVD) are shown in the context of the repeat peptide. Two TALENs must bind in an “antiparallel” manner with the appropriate spacing in order for the FokI domains to dimerize and cleave the DNA in the spacer region between the two half‐sites. “N” and “C” indicate N‐terminus and C‐terminus of each TALEN.


Figure 5. CRISPR immune response pathway. The CRISPR‐Cas immune response occurs in three phases: adaptive, biogenesis, and targeting. During the adaptive phase, short PAM containing DNA sequences (protospacers) from invading virus are recognized and integrated into the bacterial genome within the CRISPR array as spacers. The CRISPR array contains several spacers separated by short direct repeat sequences, and is located within the CRISPR locus downstream of several CRISPR associated (Cas) genes. During the biogenesis phase, the spacers are transcribed and processed into mature crRNA. In the targeting phase, crRNAs complex with Cas. This RNA‐Cas complex is guided by the crRNA to specifically bind and cleave a complimentary portion of target DNA, creating a double‐strand break (DSB) that silences the gene and prevents infection.


Figure 6. CRISPR‐Cas9 CRISPR RNA (crRNA) and single‐guide RNA (sgRNA) bound to their DNA target sites. CRISPR‐Cas9 nucleases are comprised of a Cas9 nuclease complexed with a guide RNA. This guide can either be (A) a sequence‐specific crRNA (purple) and a general tracrRNA (red) or (B) a single gRNA (sgRNA) developed by truncation and fusion of crRNA and tracrRNA sequences. The Cas9 protein has two lobes, REC and NUC, and two domains with helicase and nuclease activity: the HNH domain, which interacts with the target strand of DNA, and the RuvC domain, which interacts with the nontarget strand. The DNA target site has 20 nucleotides that are complementary to the first 20 nucleotides of the crRNA/sgRNA. The PAM site (orange) is found on the nontarget genomic DNA strand immediately after the target site.


Figure 7. Comparison of Cas proteins with distinct mechanisms. (A) Cas9 binds to genomic DNA complementary to its guide RNA (crRNA:tracrRNA duplex depicted) and cleaves the target site DNA in between the third and fourth base pair from its 5′‐NGG‐3′ protospacer adjacent motif (PAM), resulting in blunt ends at the double‐strand break. (B) The crRNA that binds to Cas12a contains a stem loop secondary structure at its 5' end. Cas12a binds to genomic DNA complementary to its crRNA and cleaves the complementary strand of the target site in between the 23rd and 24th nucleotide (nt) away from its T‐rich PAM, and between the 18th and 19th nt away from the PAM on the non‐complementary strand, resulting in 5 nt staggered overhangs. (C) Cas13a binds to mRNA complementary to its crRNA and cleaves the mRNA outside of the target site. Cas13a requires a protospacer flanking sequence (PFS) of A, U, or C.


Figure 8. Double‐strand break repair pathways used for genome editing. The two main repair pathways used by the cell to repair double‐strand breaks (DSBs) are nonhomologous end joining (NHEJ) and homology‐directed repair (HDR). NHEJ, the preferred repair pathway, is designed to repair DSBs with precise end joining; however, targeted nuclease activity can introduce direct ligation of an exogenous sequence, or insertions or deletions at the cleavage site, resulting in gene disruption. Simultaneous DSBs introduced at two nearby sites can induce large deletions or inversions, and simultaneous DSBs introduced at two sites on different chromosomes can induce translocations. HDR naturally repairs DSBs using a sister chromatid as a repair template. Delivery of an exogenous donor template with homology flanking the target site, along with a targeted nuclease, can introduce precise nucleotide modifications or sequence insertions via the HDR repair pathway.


Figure 9. On‐target assays for indel quantification. (A) Mismatch detection assays require PCR amplification of the target site from the genomic DNA of a cell pool or clone. These PCR amplicons are denatured at 95°C, then allowed to reanneal. Amplicons with indels that reanneal to wild‐type amplicons or amplicons with different indels will result in DNA bulges that are recognized and cleaved by a mismatch cleavage enzyme. Cleavage products can be visualized and quantified via gel electrophoresis. (B) Computational analysis of Sanger sequencing via TIDE or ICE also begins with PCR amplification of the target site. PCR amplicons are then analyzed by Sanger senquencing. Computational analysis is used to compare the edited pool sequence to the wild‐type sequence and quantify the editing activity. (C) Indel detection by amplicon analysis uses a fluorescent tri‐primer system to fluorescently label the PCR amplicons. Capillary electrophoresis is used to separate the fluorescent amplicons based on size, and the migration time and fluorescent intensity is used to quantify the size and relative concentration of indels. (D) Fluorescent reporter plasmids can be delivered along with targeted nuclease reagents to estimate gRNA activity. One such plasmid contains the sequence for GFP and RFP, with the target site in between such that the RFP sequence is out of frame, expressing the GFP but not the RFP protein. Cleavage at the target site will induce indels in between the two fluorescent proteins, some of which will cause a frameshift allowing expression of RFP. Cleavage and editing can be measured via RFP and GFP fluorescence intensity. (E) Another fluorescent reporter plasmid contains the sequence for a fluorescent protein that is disrupted by the target site flanked by direct repeat sequences (DR). Cleavage at the target site induces repair via the SSA pathway, which restores the fluorescent protein expression. (F) High‐resolution melting analysis DNA dependent fluorescent dye is allowed to bind to target site DNA amplicons. Fluorescence intensity is measured as the amplicons are slowly denatured over time. Amplicons with indels will denature at different rates than the wild‐type amplicons.


Figure 10. On‐Target assays for confirming desired editing events. (A) The restriction fragment polymorphism (RFLP) assay confirms successful knockin by introducing a new restriction enzyme site along with the desired mutation. After PCR amplification of the target site, the DNA is incubated with the restriction enzyme. The restriction enzyme will only cleave amplicons with successful integration and introduction of the restriction site. The cleavage products can be visualized via gel electrophoresis. The wild‐type amplicon and the cleavage products will be present for heterozygous knockins, and the cleavage products alone will be present in clones with homozygous knockin alleles. (B) Junction PCRs can be used to confirm the successful integration of the desired modification. Primers are designed to amplify the junctions between the genomic DNA and the integrated sequence. The presence of an amplicon of the correct size for both junctions confirms a successful knockin event. (C) Western blot or ELISA can be used to confirm the successful knockin of a mutation or sequence integration that is recognizable by an antibody, such as a mutation that causes a structural change or the addition of a protein domain. (D) Where appropriate, a fluorescent tag can be included along with the desired mutation, which can be used to detect successful integration.


Figure 11. On‐target assays for indel quantification and confirming desired editing events (knockin and knockout). (A) Computational analysis of Sanger sequencing via TIDER or ICE begins with PCR amplification of the target site. PCR amplicons are then analyzed by Sanger sequencing. Computational analysis is used to compare the edited pool sequence to the wild‐type sequence and quantify the editing activity. TIDER requires comparison to a positive control sequence for analysis of HDR, while ICE does not. (B) For NHEJ and HDR analysis via targeted genome sequencing, edited genomic DNA is first PCR amplified using site‐specific primers with adaptors, then PCR amplified using indexing primers. These amplicons are sequenced using next‐generation sequencing, and both NHEJ and HDR events can be analyzed using the same data set.


Figure 12. Modifications to the CRISPR‐Cas9 platform to reduce off‐target editing. (A) Modifications made to the gRNA to increase specificity. Tru‐sgRNAs are truncated gRNAs with complementarity lengths of 17 or 18 nucleotides. GG‐sgRNAs have two target site independent, 5’ guanine residues, which have been shown to increase sgRNA specificity. (B) Alternative Cas9 architectures to reduce off‐target cleavage. Nickases contain a point mutation that renders one of the nuclease domains inactive (indicated with gray oval) resulting in the creation of a single‐strand DNA nick. Paired nickases are depicted in the PAM‐out orientation. With proper orientation and gRNA offset, paired nickases create two single‐stranded DNA nicks, which can result in a DSB with staggered overhangs. RNA‐guided FokI nucleases (RFNs) are composed of catalytically dead Cas9 (dCas9) fused to the FokI nuclease domain. The FokI nuclease requires dimerization for cleavage. DSBs are created between RFNs with proper orientation and spacing.


Figure 13. CRISPR epigenetic regulators. (A) dCas9 can repress gene expression by blocking transcription initiation or transcription elongation. Fusion of a Krüppel‐associated box (KRAB) domain to the dCas9 protein improves this repressive activity in mammalian cells. CRISPR activators can generally be categorized into dCas9 protein fusion (B) or gRNA aptamer fusion strategies (C). (B) VP64 (VP16 × 4) and VPR (VP64, p65, and Rta domains in tandem) have been fused directly to dCas9 for use as a gene activator. The SunTag system fuses several GCN4 peptides to the dCas9 protein. GCN4 peptides are recognized by a GCN4 scFv antibody fused to a VP64 domain. (C) Replacing the tetraloop and stemloop of the gRNA with MS2 aptamers allows recruitment of tandem p65‐HSF1 domains fused to MCP, which binds to the MS2 aptamer. This was termed the synergistic activation mediator (SAM) system. The Casilio system appends several PBS domains to the 3’ end of the gRNA. VP64 domains are recruited to the gRNA via fusion to a Pumilio/FBF (PUF) RNA‐binding domain, which binds to the corresponding PUF binding site (PBS). The male‐specific bacteriophage‐2 (MS2) aptamer or the com aptamer have also been appended to the 3’ end of the gRNA. VP64 is recruited to the MS2 domain and KRAB fused to COM is recruited to the com aptamer. This system was used to create orthogonal CRISPR transcriptional regulators for simultaneous gene activation and repression.


Figure 14. Pooled and arrayed screening strategies. (A) A pooled screen is typically conducted using lentiviral particles. The library pool is delivered to cells with and without Cas9 expression. A selective pressure is applied to the cells, and next‐generation sequencing is used to analyze the gRNA sequences that have been enriched (positive selection) or depleted (negative selection). (B) In an arrayed screen, gRNAs can be delivered as RNA, plasmid, or lentivirus. Cas9 expressing cells are plated into 96‐ or 384‐well format, and each gRNA in the library is delivered into an individual well. Wells in which the desired phenotype occurs can then be easily traced back to the gRNA and targeted gene.


Figure 15. Cell line creation workflow. Steps involved in using CRISPR‐Cas9 to develop a targeted genome‐modified cell line. Reagents are designed and validated (1‐5), delivered to cells (6), screened for desired modification(s) (7‐9), and the final genetically modified clone is expanded, genotype confirmed, and cryopreserved (10). The funnel depicts the large number of input cells (orange) with a rare genome edit (green), which is isolated and expanded to form the cell line of interest.


Figure 16. Animal model creation workflow. Steps involved in using CRISPR‐Cas9 to develop a targeted genome‐modified animal model. Reagents are designed and validated (1‐5), injected into fertilized oocytes (6), transferred to a pseudopregnant female (7), and after birth (8), the offspring are screened for the desired modification(s) (9). Selected animals must be bred to confirm germline transmission of the genome edits and to establish the line (10).


Figure 17. Using programmable nucleases for human ex vivo therapy. For human ex vivo therapy using programmable nucleases, target cells are (A) harvested from a patient and (B) expanded in culture. (C) Nuclease(s) and donor DNA, (if required) are delivered to patient cells to allow for the desired genomic modification. Donor DNA can be delivered via plasmid, single stranded DNA, single stranded oligonucleotide donor, or virus. (D) Genetically modified cells are then reintroduced into the patient (autologous transplant) and need to home to the appropriate niche(s) and expand to provide a therapeutic effect.
References
 1. Aach J , Mali P , Church GM . CasFinder: Flexible algorithm for identifying specific Cas9 targets in genomes. bioRxiv 2014. doi: 10.1101/005074.
 2. Abadi S , Yan WX , Amar D , Mayrose I . A machine learning approach for predicting CRISPR‐Cas9 cleavage efficiencies and patterns underlying its mechanism of action. PLoS Comput Biol 13: 1‐24, 2017.
 3. Abudayyeh OO , Gootenberg JS , Essletzbichler P , Han S , Joung J , Belanto JJ , Verdine V , Cox DBT , Kellner MJ , Regev A , Lander ES , Voytas DF , Ting AY , Zhang F . RNA targeting with CRISPR‐Cas13. Nature 550: 280‐284, 2017.
 4. Abudayyeh OO , Gootenberg JS , Konermann S , Joung J , Slaymaker IM , Cox DBT , Shmakov S , Makarova KS , Semenova E , Minakhin L , Severinov K , Regev A , Lander ES , Koonin EV , Zhang F. C2c2 is a single‐component programmable RNA‐guided RNA‐targeting CRISPR effector. Science 353: aaf5573, 2016.
 5. Alzubi J , Pallant C , Mussolino C , Howe SJ , Thrasher AJ , Cathomen T . Targeted genome editing restores T cell differentiation in a humanized X‐SCID pluripotent stem cell disease model. Sci Rep 7: 2017.
 6. Anders C , Bargsten K , Jinek M . Structural plasticity of PAM recognition by engineered variants of the RNA‐guided endonuclease Cas9. Mol Cell 61: 895‐902, 2016.
 7. Anders C , Niewoehner O , Duerst A , Jinek M . Structural basis of PAM‐dependent target DNA recognition by the Cas9 endonuclease. Nature 513: 569‐573, 2014.
 8. Argast GM , Stephens KM , Emond MJ , Monnat RJ . I‐PpoI and I‐CreI homing site sequence degeneracy determined by random mutagenesis and sequential in vitro enrichment. J Mol Biol 280: 345‐353, 1998.
 9. Arnould S , Chames P , Perez C , Lacroix E , Duclert A , Epinat JC , Stricher F , Petit AS , Patin A , Guillier S , Rolland S , Prieto J , Blanco FJ , Bravo J , Montoya G , Serrano L , Duchateau P , Pâques F . Engineering of large numbers of highly specific homing endonucleases that induce recombination on novel DNA targets. J Mol Biol 355: 443‐458, 2006.
 10. Bae KH , Kwon YD , Shin HC , Hwang MS , Ryu EH , Park KS , Yang HY , Lee DK , Lee Y , Park J , Kwon HS , Kim HW , Yeh BI , Lee HW , Sohn SH , Yoon J , Seol W , Kim JS . Human zinc fingers as building blocks in the construction of artificial transcription factors. Nat Biotechnol 21: 275‐280, 2003.
 11. Bae S , Kweon J , Kim HS , Kim JS . Microhomology‐based choice of Cas9 nuclease target sites. Nat Methods 11: 705‐706, 2014.
 12. Bae S , Park J , Kim JS . Cas‐OFFinder: A fast and versatile algorithm that searches for potential off‐target sites of Cas9 RNA‐guided endonucleases. Bioinformatics 30: 1473‐1475, 2014.
 13. Bak RO , Porteus MH . CRISPR‐mediated integration of large gene cassettes using AAV donor vectors. Cell Rep 20: 750‐756, 2017.
 14. Baker KE , Parker R. Nonsense‐mediated mRNA decay: Terminating erroneous gene expression. Curr Opin Cell Biol 16: 293‐299, 2004.
 15. Baltimore D , Berg P , Botchan M , Carroll D , Charo RA , Church G , Corn JE , Daley GQ , Doudna JA , Fenner M , Greely HT , Jinek M , Martin GS , Penhoet E , Puck J , Sternberg SH , Weissman JS , Yamamoto KR . A prudent path forward for genomic engineering and germline gene modification. Science 348: 36‐38, 2015.
 16. Barrangou R , Marraffini LA . CRISPR‐Cas systems: Prokaryotes upgrade to adaptive immunity. Mol Cell 54: 234‐244, 2014.
 17. Beerli RR , Barbas CF . Engineering polydactyl zinc‐finger transcription factors. Nat Biotechnol 20: 135‐141, 2002.
 18. Beerli RR , Segal DJ , Dreier B , Barbas CF . Toward controlling gene expression at will: Specific regulation of the erbB‐2/HER‐2 promoter by using polydactyl zinc finger proteins constructed from modular building blocks. Proc Natl Acad Sci 95: 14628‐14633, 1998.
 19. Bell CC , Magor GW , Gillinder KR , Perkins AC . A high‐throughput screening strategy for detecting CRISPR‐Cas9 induced mutations using next‐generation sequencing. BMC Genomics 15: 1002, 2014.
 20. Bestor TH . Gene silencing as a threat to the success of gene therapy. J Clin Invest 105: 409‐411, 2000.
 21. Bétermier M , Bertrand P , Lopez BS . Is non‐homologous end‐joining really an inherently error‐prone process? PLoS Genet 10: e1004086, 2014.
 22. Beylot B , Spassky A . Chemical probing shows that the intron‐encoded endonuclease I‐SceI distorts DNA through binding in monomeric form to its homing site. J Biol Chem 276: 25243‐25253, 2001.
 23. Bibikova M , Beumer K , Trautman JK , Carroll D . Enhancing gene targeting with designed zinc finger nucleases. Science 300: 764, 2003.
 24. Bibikova M , Golic M , Golic KG , Carroll D . Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc‐finger nucleases. Genetics 161: 1169‐1175, 2002.
 25. Bikard D , Jiang W , Samai P , Hochschild A , Zhang F , Marraffini LA . Programmable repression and activation of bacterial gene expression using an engineered CRISPR‐Cas system. Nucleic Acids Res 41: 7429‐7437, 2013.
 26. Biswas A , Gagnon JN , Brouns SJJ , Fineran PC , Brown CM . CRISPRTarget: Bioinformatic prediction and analysis of crRNA targets. RNA Biol 10: 817‐827, 2013.
 27. Bjurström CF , Mojadidi M , Phillips J , Kuo C , Lai S , Lill GR , Cooper A , Kaufman M , Urbinati F , Wang X , Hollis RP , Kohn DB . Reactivating fetal hemoglobin expression in human adult erythroblasts through BCL11A knockdown using targeted endonucleases. Mol Ther Nucleic Acids 5: e351, 2016.
 28. Black JB , Adler AF , Wang HG , D'Ippolito AM , Hutchinson HA , Reddy TE , Pitt GS , Leong KW , Gersbach CA . Targeted epigenetic remodeling of endogenous loci by CRISPR/Cas9‐based transcriptional activators directly converts fibroblasts to neuronal cells. Cell Stem Cell 19: 406‐414, 2016.
 29. Blasco RB , Karaca E , Ambrogio C , Cheong TC , Karayol E , Minero VG , Voena C , Chiarle R . Simple and rapid in vivo generation of chromosomal rearrangements using CRISPR/Cas9 technology. Cell Rep 9: 1219‐1227, 2014.
 30. Blin K , Pedersen LE , Weber T , Lee SY . CRISPy‐web: An online resource to design sgRNAs for CRISPR applications. Synth Syst Biotechnol 1: 118‐121, 2016.
 31. Boch J , Bonas U . Xanthomonas AvrBs3 family‐type III effectors: Discovery and function. Annu Rev Phytopathol 48: 419‐436, 2010.
 32. Boch J , Scholze H , Schornack S , Landgraf A , Hahn S , Kay S , Lahaye T , Nickstadt A , Bonas U . Breaking the code of DNA binding specificity of TAL‐type III effectors. Science 326: 1509‐1512, 2009.
 33. Bogdanove AJ , Voytas DF . TAL effectors: Customizable proteins for DNA targeting. Science 333: 1843‐1846, 2011.
 34. Boissel S , Jarjour J , Astrakhan A , Adey A , Gouble A , Duchateau P , Shendure J , Stoddard BL , Certo MT , Baker D , Scharenberg AM . MegaTALs: A rare‐cleaving nuclease architecture for therapeutic genome engineering. Nucleic Acids Res 42: 2591‐2601, 2014.
 35. Bondy‐Denomy J , Pawluk A , Maxwell KL , Davidson AR . Bacteriophage genes that inactivate the CRISPR/Cas bacterial immune system. Nature 493: 429‐432, 2013.
 36. Bousso P , Wahn V , Douagi I , Horneff G , Pannetier C , Le Deist F , Zepp F , Niehues T , Kourilsky P , Fischer A , de Saint Basile G . Diversity, functionality, and stability of the T cell repertoire derived in vivo from a single human T cell precursor. Proc Natl Acad Sci U S A 97: 274‐278, 2000.
 37. Brinkman EK , Chen T , Amendola M , van Steensel B . Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res 42: e168, 2014.
 38. Brinkman EK , Kousholt AN , Harmsen T , Leemans C , Chen T , Jonkers J , van Steensel B . Easy quantification of template‐directed CRISPR/Cas9 editing. Nucleic Acids Res 46: e58, 2017.
 39. Buckley RH , Schiff SE , Schiff RI , Markert L , Williams LW , Roberts JL , Myers LA , Ward FE . Hematopoietic stem‐cell transplantation for the treatment of severe combined immunodeficiency. N Engl J Med 340: 508‐516, 1999.
 40. Canela A , Sridharan S , Sciascia N , Tubbs A , Meltzer P , Sleckman BP , Nussenzweig A . DNA breaks and end resection measured genome‐wide by end sequencing. Mol Cell 63: 898‐911, 2016.
 41. Carlson DF , Lancto CA , Zang B , Kim ES , Walton M , Oldeschulte D , Seabury C , Sonstegard TS , Fahrenkrug SC . Production of hornless dairy cattle from genome‐edited cell lines. Nat Biotechnol 34: 479‐481, 2016.
 42. Carroll D . Genome Engineering with Targetable Nucleases. Annu Rev Biochem 83: 409‐439, 2014.
 43. Carter‐Johnson J . Defining the spectrum of “normal”: What is a disease? [Online]. Cent. Ethics Humanit. Life Sci. Michigan State Univ.: 2016. https://msubioethics.com/2016/10/20/defining‐the‐spectrum‐of‐normal/ [accessed 4 April 2018].
 44. Cavazzana‐Calvo M , Hacein‐Bey S , De Saint Basile G , Gross F , Yvon E , Nusbaum P , Selz F , Hue C , Certain S , Casanova JL , Bousso P , Le Deist F , Fischer A . Gene therapy of human severe combined immunodeficiency (SCID)‐X1 disease. Science 288: 669‐672, 2000.
 45. Cermak T , Doyle EL , Christian M , Wang L , Zhang Y , Schmidt C , Baller JA , Somia N V. , Bogdanove AJ, Voytas DF. Efficient design and assembly of custom TALEN and other TAL effector‐based constructs for DNA targeting. Nucleic Acids Res. 39: e82, 2011.
 46. Chakraborty S , Ji H , Kabadi AM , Gersbach CA , Christoforou N , Leong KW . A CRISPR/Cas9‐based system for reprogramming cell lineage specification. Stem Cell Reports 3: 940‐947, 2014.
 47. Chapman JR , Taylor MRG , Boulton SJ . Playing the end game: DNA double‐strand break repair pathway choice. Mol Cell 47: 497‐510, 2012.
 48. Chari R , Yeo NC , Chavez A , Church GM . SgRNA Scorer 2.0: A species‐independent model to predict CRISPR/Cas9 activity. ACS Synth Biol 6: 902‐904, 2017.
 49. Charo RA , Hynes RO , Beier DW , Clayton EW , Coller BS , Evans JH , Izpisua Belmonte JC , Jaenisch R , Kahn J , Levy‐Lahad E , Lovell‐Badge R , Marchant G , Merchant J , Naldini L , Pei D , Porteus M , Rossant J , Scheufele DA , Serageldin I , Terry S , Weissman J , Yamamoto KR , Bowman KW , Gonzalez M , Roberts JR , Pope AM , Sharples FE . Human genome editing: Science, ethics and governance. Natl Acad Sci 2017. doi: 10.17226/24623.
 50. Chavez A , Scheiman J , Vora S , Pruitt BW , Tuttle M , Iyer EPR , Lin S , Kiani S , Guzman CD , Wiegand DJ , Ter‐Ovanesyan D , Braff JL , Davidsohn N , Housden BE , Perrimon N , Weiss R , Aach J , Collins JJ , Church GM . Highly‐efficient Cas9‐mediated transcriptional programming. Nat Methods 12: 326‐328, 2015.
 51. Chen B , Gilbert LA , Cimini BA , Schnitzbauer J , Zhang W , Li G , Park J , Blackburn EH , Weissman JS , Qi LS , Huang B . Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 155: 1479‐1491, 2013.
 52. Chen F , Ding X , Feng Y , Seebeck T , Jiang Y , Davis GD . Targeted activation of diverse CRISPR‐Cas systems for mammalian genome editing via proximal CRISPR targeting. Nat Commun 8: 1‐12, 2017.
 53. Chen F , Pruett‐Miller SM , Davis GD . Gene editing using ssODNs with engineered endonucleases. Methods Mol Biol 1239: 251‐265, 2015.
 54. Chen F , Pruett‐Miller SM , Huang Y , Gjoka M , Duda K , Taunton J , Collingwood TN , Frodin M , Davis GD . High‐frequency genome editing using ssDNA oligonucleotides with zinc‐finger nucleases. Nat Methods 8: 753‐757, 2011.
 55. Chen JS , Dagdas YS , Kleinstiver BP , Welch MM , Sousa AA , Harrington LB , Sternberg SH , Joung JK , Yildiz A , Doudna JA . Enhanced proofreading governs CRISPR‐Cas9 targeting accuracy. Nature 550: 407‐410, 2017.
 56. Chen JS , Ma E , Harrington LB , Da Costa M , Tian X , Palefsky JM , Doudna JA . CRISPR‐Cas12a target binding unleashes indiscriminate single‐stranded DNase activity. Science 360: 436‐439, 2018.
 57. Chen S , Lee B , Lee AYF , Modzelewski AJ , He L . Highly efficient mouse genome editing by CRISPR ribonucleoprotein electroporation of zygotes. J Biol Chem 291: 14457‐14467, 2016.
 58. Cheng AW , Jillette N , Lee P , Plaskon D , Fujiwara Y , Wang W , Taghbalout A , Wang H . Casilio: A versatile CRISPR‐Cas9‐Pumilio hybrid for gene regulation and genomic labeling. Cell Res 26: 254‐257, 2016.
 59. Cheng AW , Wang H , Yang H , Shi L , Katz Y , Theunissen TW , Rangarajan S , Shivalila CS , Dadon DB , Jaenisch R . Multiplexed activation of endogenous genes by CRISPR‐on, an RNA‐guided transcriptional activator system. Cell Res 23: 1163‐1171, 2013.
 60. Chevalier BS , Kortemme T , Chadsey MS , Baker D , Monnat RJ , Stoddard BL . Design, activity, and structure of a highly specific artificial endonuclease. Mol Cell 10: 895‐905, 2002.
 61. Chevalier BS , Stoddard BL. Homing endonucleases: Structural and functional insight into the catalysts of intron/intein mobility. Nucleic Acids Res 29: 3757‐3774, 2001.
 62. Cho SW , Kim S , Kim JM , Kim JS . Targeted genome engineering in human cells with the Cas9 RNA‐guided endonuclease. Nat Biotechnol 31: 230‐232, 2013.
 63. Cho SW , Kim S , Kim Y , Kweon J , Kim HS , Bae S , Kim J‐S . Analysis of off‐target effects of CRISPR/Cas‐derived RNA‐guided endonucleases and nickases. Genome Res 24: 132‐144, 2014.
 64. Choudhury SR , Cui Y , Lubecka K , Stefanska B , Irudayaraj J . CRISPR‐dCas9 mediated TET1 targeting for selective DNA demethylation at BRCA1 promoter. Oncotarget 7: 46545‐46556, 2016.
 65. Choulika A , Perrin A , Dujon B , Nicolas J . Induction of homologous recombination in mammalian chromosomes by using the I‐SceI system of Saccharomyces cerevisiae. Mol Cell Biol 15: 1968‐1973, 1995.
 66. Christian M , Cermak T , Doyle EL , Schmidt C , Zhang F , Hummel A , Bogdanove AJ , Voytas DF . Targeting DNA double‐strand breaks with TAL effector nucleases. Genetics 186: 756‐761, 2010.
 67. Chylinski K , Le Rhun A , Charpentier E . The tracrRNA and Cas9 families of type II CRISPR‐Cas immunity systems. RNA Biol 10: 726‐737, 2013.
 68. Cleary MA , Kilian K , Wang Y , Bradshaw J , Cavet G , Ge W , Kulkarni A , Paddison PJ , Chang K , Sheth N , Leproust E , Coffey EM , Burchard J , McCombie WR , Linsley P , Hannon GJ . Production of complex nucleic acid libraries using highly parallel in situ oligonucleotide synthesis. Nat Methods 1: 241‐248, 2004.
 69. Cong L , Ran FA , Cox D , Lin S , Barretto R , Habib N , Hsu PD , Wu X , Jiang W , Marraffini LA , Zhang F . Multiplex genome engineering using CRISPR/Cas systems. Science 339: 819‐823, 2013.
 70. Cradick TJ , Qiu P , Lee CM , Fine EJ , Bao G . COSMID: A web‐based tool for identifying and validating CRISPR/Cas off‐target sites. Mol Ther Nucleic Acids 3, 2014.
 71. Crosetto N , Mitra A , Silva MJ , Bienko M , Dojer N , Wang Q , Karaca E , Chiarle R , Skrzypczak M , Ginalski K , Pasero P , Rowicka M , Dikic I . Nucleotide‐resolution DNA double‐strand break mapping by next‐generation sequencing. Nat Methods 10: 361‐365, 2013.
 72. Daboussi F , Zaslavskiy M , Poirot L , Loperfido M , Gouble A , Guyot V , Leduc S , Galetto R , Grizot S , Oficjalska D , Perez C , Delacôte F , Dupuy A , Chion‐Sotinel I , Le Clerre D , Lebuhotel C , Danos O , Lemaire F , Oussedik K , Cédrone F , Epinat JC , Smith J , Yáñez‐Muñoz RJ , Dickson G , Popplewell L , Koo T , Vandendriessche T , Chuah MK , Duclert A , Duchateau P , Pâques F . Chromosomal context and epigenetic mechanisms control the efficacy of genome editing by rare‐cutting designer endonucleases. Nucleic Acids Res 40: 6367‐6379, 2012.
 73. Dellinger DJ , Timár Z , Myerson J , Sierzchala AB , Turner J , Ferreira F , Kupihár Z , Dellinger G , Hill KW , Powell JA , Sampson JR , Caruthers MH . Streamlined process for the chemical synthesis of RNA using 2′‐O‐thionocarbamate‐protected nucleoside phosphoramidites in the solid phase. J Am Chem Soc 133: 11540‐11556, 2011.
 74. Deng D , Yan C , Pan X , Mahfouz M , Wang J , Zhu J‐K , Shi Y , Yan N . Structural basis for sequence‐specific recognition of DNA by TAL effectors. Science 335: 720‐723, 2012.
 75. Denner J . Advances in organ transplant from pigs. Science 357: 1238‐1239, 2017.
 76. Dever DP , Bak RO , Reinisch A , Camarena J , Washington G , Nicolas CE , Pavel‐Dinu M , Saxena N , Wilkens AB , Mantri S , Uchida N , Hendel A , Narla A , Majeti R , Weinberg KI , Porteus MH . CRISPR/Cas9 β‐globin gene targeting in human haematopoietic stem cells. Nature 539: 384‐389, 2016.
 77. DeWitt MA , Magis W , Bray NL , Wang T , Berman JR , Urbinati F , Heo SJ , Mitros T , Muñoz DP , Boffelli D , Kohn DB , Walters MC , Carroll D , Martin DIK , Corn JE . Selection‐free genome editing of the sickle mutation in human adult hematopoietic stem/progenitor cells. Sci Transl Med 8: 360ra134, 2016.
 78. DiGiusto DL , Cannon PM , Holmes MC , Li L , Rao A , Wang J , Lee G , Gregory PD , Kim KA , Hayward SB , Meyer K , Exline C , Lopez E , Henley J , Gonzalez N , Bedell V , Stan R , Zaia JA . Preclinical development and qualification of ZFN‐mediated CCR5 disruption in human hematopoietic stem/progenitor cells. Mol Ther Methods Clin Dev 3: 16067, 2016.
 79. Doench JG , Fusi N , Sullender M , Hegde M , Vaimberg EW , Donovan KF , Smith I , Tothova Z , Wilen C , Orchard R , Virgin HW , Listgarten J , Root DE . Optimized sgRNA design to maximize activity and minimize off‐target effects of CRISPR‐Cas9. Nat Biotechnol 34: 184‐191, 2016.
 80. Doench JG , Hartenian E , Graham DB , Tothova Z , Hegde M , Smith I , Sullender M , Ebert BL , Xavier RJ , Root DE . Rational design of highly active sgRNAs for CRISPR‐Cas9‐mediated gene inactivation. Nat Biotechnol 32: 1262‐1267, 2014.
 81. Dominguez AA , Lim WA , Qi LS . Beyond editing: Repurposing CRISPR–Cas9 for precision genome regulation and interrogation. Nat Rev Mol Cell Biol 17: 5‐15, 2016.
 82. Donoho G , Jasin M , Berg P . Analysis of gene targeting and intrachromosomal homologous recombination stimulated by genomic double‐strand breaks in mouse embryonic stem cells. Mol Cell Biol 18: 4070‐4078, 1998.
 83. Doyle EL , Hummel AW , Demorest ZL , Starker CG , Voytas DF , Bradley P , Bogdanove AJ . TAL effector specificity for base 0 of the DNA target is altered in a complex, effector‐ and assay‐dependent manner by substitutions for the tryptophan in cryptic repeat ‐1. PLoS One 8(12): 2013.
 84. Doyon Y , McCammon JM , Miller JC , Faraji F , Ngo C , Katibah GE , Amora R , Hocking TD , Zhang L , Rebar EJ , Gregory PD , Urnov FD , Amacher SL . Heritable targeted gene disruption in zebrafish using designed zinc‐finger nucleases. Nat Biotechnol 26: 702‐708, 2008.
 85. Duncan BK , Miller JH. Mutagenic deamination of cytosine residues in DNA. Nature 287: 560‐561, 1980.
 86. Dykxhoorn DM , Lieberman J. The silent revolution: RNA interference as basic biology, research tool, and therapeutic. Annu Rev Med 56: 401‐423, 2005.
 87. East‐Seletsky A , O'Connell MR , Knight SC , Burstein D , Cate JHD , Tjian R , Doudna JA . Two distinct RNase activities of CRISPR‐C2c2 enable guide‐RNA processing and RNA detection. Nature 538: 270‐273, 2016.
 88. Egli D , Zuccaro M , Kosicki M , Church G , Bradley A , Jasin M . Inter‐homologue repair in fertilized human eggs? Nature 560: E5‐E7, 2018. doi: https://doi.org/10.1101/181255.
 89. Ehrke‐Schulz E , Schiwon M , Leitner T , Dávid S , Bergmann T , Liu J , Ehrhardt A . CRISPR/Cas9 delivery with one single adenoviral vector devoid of all viral genes. Sci Rep 7: 1‐11, 2017.
 90. Elliott B , Richardson C , Winderbaum J , Nickoloff JA , Jasin M . Gene conversion tracts from double‐strand break repair in mammalian cells. Mol Cell Biol 18: 93‐101, 1998.
 91. Ellis BL , Hirsch ML , Barker JC , Connelly JP , Steininger RJ , Porteus MH . A survey of ex vivo/in vitro transduction efficiency of mammalian primary cells and cell lines with Nine natural adeno‐associated virus (AAV1‐9) and one engineered adeno‐associated virus serotype. Virol. J. 10: 2013.
 92. Engler C , Gruetzner R , Kandzia R , Marillonnet S . Golden gate shuffling: A one‐pot DNA shuffling method based on type IIs restriction enzymes. PLoS One 4: e5553, 2009.
 93. Esvelt KM , Mali P , Braff JL , Moosburne M , Yaung SJ , Church GM . Orthogonal Cas9 proteins for RNA‐guided gene regulation and editing. Nat Methods 10: 1116‐1121, 2013.
 94. Farzadfard F , Perli SD , Lu TK . Tunable and multifunctional eukaryotic transcription factors based on CRISPR/Cas. ACS Synth Biol 2: 604‐613, 2013.
 95. Fineran PC , Charpentier E . Memory of viral infections by CRISPR‐Cas adaptive immune systems: Acquisition of new information. Virology 434: 202‐209, 2012.
 96. Fischer A , Le Deist F , Hacein‐Bey‐Abina S , André‐Schmutz I , De Saint Basile G , De Villartay JP , Cavazzana‐Calvo M . Severe combined immunodeficiency. A model disease for molecular immunology and therapy. Immunol Rev. 203: 98‐109, 2005.
 97. Flick KE , Jurica MS , Monnat RJ , Stoddard BL . DNA binding and cleavage by the nuclear intron‐encoded homing endonuclease I‐Ppol. Nature 394: 96‐101, 1998.
 98. Fonfara I , Le Rhun A , Chylinski K , Makarova KS , Lécrivain AL , Bzdrenga J , Koonin E V. , Charpentier E. Phylogeny of Cas9 determines functional exchangeability of dual‐RNA and Cas9 among orthologous type II CRISPR‐Cas systems. Nucleic Acids Res 42: 2577‐2590, 2014.
 99. Friedland AE , Baral R , Singhal P , Loveluck K , Shen S , Sanchez M , Marco E , Gotta GM , Maeder ML , Kennedy EM , Kornepati AVR , Sousa A , Collins MA , Jayaram H , Cullen BR , Bumcrot D . Characterization of Staphylococcus aureus Cas9: A smaller Cas9 for all‐in‐one adeno‐associated virus delivery and paired nickase applications. Genome Biol 16, 2015.
 100. Frischmeyer PA , Dietz HC . Nonsense‐mediated mRNA decay in health and disease. Hum Mol Genet 8: 1893‐1900, 1999.
 101. Frock RL , Hu J , Meyers RM , Ho YJ , Kii E , Alt FW . Genome‐wide detection of DNA double‐stranded breaks induced by engineered nucleases. Nat Biotechnol 33: 179‐188, 2015.
 102. Fu Y , Foden JA , Khayter C , Maeder ML , Reyon D , Joung JK , Sander JD . High‐frequency off‐target mutagenesis induced by CRISPR‐Cas nucleases in human cells. Nat Biotechnol 31: 822‐826, 2013.
 103. Fu Y , Sander JD , Reyon D , Cascio VM , Joung JK . Improving CRISPR‐Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol 32: 279‐284, 2014.
 104. Fujihara Y , Ikawa M . CRISPR/Cas9‐based genome editing in mice by single plasmid injection. Meth Enzymol 546: 319‐336, 2014.
 105. Fulco CP , Munschauer M , Anyoha R , Munson G , Sharon R , Perez EM , Kane M , Cleary B , Lander ES , Engreitz JM . Systematic mapping of functional enhancer–promoter connections with CRISPR interference. Science 354: 769‐773, 2016.
 106. Gabriel R , Lombardo A , Arens A , Miller JC , Genovese P , Kaeppel C , Nowrouzi A , Bartholomae CC , Wang J , Friedman G , Holmes MC , Gregory PD , Glimm H , Schmidt M , Naldini L , Von Kalle C . An unbiased genome‐wide analysis of zinc‐finger nuclease specificity. Nat Biotechnol 29: 816‐823, 2011.
 107. Gaj T , Epstein BE , Schaffer D V . Genome engineering using Adeno‐associated virus: Basic and clinical research applications. Mol. Ther 24: 458‐464, 2016.
 108. Gaj T , Staahl BT , Rodrigues GMC , Limsirichai P , Ekman FK , Doudna JA , Schaffer DV . Targeted gene knock‐in by homology‐directed genome editing using Cas9 ribonucleoprotein and AAV donor delivery. Nucleic Acids Res 45, 2017.
 109. Galvani AP , Novembre J . The evolutionary history of the CCR5‐Δ32 HIV‐resistance mutation. Microbes Infect 7: 301‐308, 2005.
 110. Gao X , Tao Y , Lamas V , Huang M , Yeh WH , Pan B , Hu YJ , Hu JH , Thompson DB , Shu Y , Li Y , Wang H , Yang S , Xu Q , Polley DB , Liberman MC , Kong WJ , Holt JR , Chen ZY , Liu DR . Treatment of autosomal dominant hearing loss by in vivo delivery of genome editing agents. Nature 553: 217‐221, 2018.
 111. Gao X , Tsang JCH , Gaba F , Wu D , Lu L , Liu P . Comparison of TALE designer transcription factors and the CRISPR/dCas9 in regulation of gene expression by targeting enhancers. Nucleic Acids Res 42: e155, 2014.
 112. Gao Y , Xiong X , Wong S , Charles EJ , Lim WA , Qi LS . Complex transcriptional modulation with orthogonal and inducible dCas9 regulators. Nat Methods 13: 1043‐1049, 2016.
 113. Garneau JE , Dupuis MÈ , Villion M , Romero DA , Barrangou R , Boyaval P , Fremaux C , Horvath P , Magadán AH , Moineau S . The CRISPR/cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468: 67‐71, 2010.
 114. Gasiunas G , Barrangou R , Horvath P , Siksnys V . Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc NATL Acad Sci U S A 109: E2579‐E2586, 2012.
 115. Gaspar HB , Cooray S , Gilmour KC , Parsley KL , Adams S , Howe SJ , Al Ghonaium A , Bayford J , Brown L , Davies EG , Kinnon C , Thrasher AJ . Long‐term persistence of a polyclonal T cell repertoire after gene therapy for X‐linked severe combined immunodeficiency. Sci Transl Med 3: 97ra79, 2011.
 116. Gasperini M , Findlay GM , McKenna A , Milbank JH , Lee C , Zhang MD , Cusanovich DA , Shendure J . CRISPR/Cas9‐mediated scanning for regulatory elements required for HPRT1 expression via thousands of large, programmed genomic deletions. Am J Hum Genet 101: 192‐205, 2017.
 117. Gaudelli NM , Komor AC , Rees HA , Packer MS , Badran AH , Bryson DI , Liu DR . Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551: 464‐471, 2017.
 118. Gerhke JM , Cervantes OR , Clement MK , Pinello L , Joung JK . High‐precision CRISPR‐Cas9 base editors with minimized bystander and off‐target mutations. bioRxiv 2018. doi: 10.1101/273938.
 119. Geurts AM , Cost GJ , Freyvert Y , Zeitler B , Miller JC , Choi VM , Jenkins SS , Wood A , Cui X , Meng X , Vincent A , Lam S , Michalkiewicz M , Schilling R , Foeckler J , Kalloway S , Weiler H , Ménoret S , Anegon I , Davis GD , Zhang L , Rebar EJ , Gregory PD , Urnov FD , Jacob HJ , Buelow R . Knockout rats via embryo microinjection of zinc‐finger nucleases. Science 325: 433, 2009.
 120. Gilbert LA , Horlbeck MA , Adamson B , Villalta JE , Chen Y , Whitehead EH , Guimaraes C , Panning B , Ploegh HL , Bassik MC , Qi LS , Kampmann M , Weissman JS . Genome‐scale CRISPR‐mediated control of gene repression and activation. Cell 159: 647‐661, 2014.
 121. Gilbert LA , Larson MH , Morsut L , Liu Z , Brar GA , Torres SE , Stern‐Ginossar N , Brandman O , Whitehead EH , Doudna JA , Lim WA , Weissman JS , Qi LS . CRISPR‐mediated modular RNA‐guided regulation of transcription in eukaryotes. Cell 154: 442‐451, 2013.
 122. Glass WG , McDermott DH , Lim JK , Lekhong S , Yu SF , Frank WA , Pape J , Cheshier RC , Murphy PM . CCR5 deficiency increases risk of symptomatic West Nile virus infection. J Exp Med 203: 35‐40, 2006.
 123. Goodarzi AA , Jeggo PA . The repair and signaling responses to DNA double‐strand breaks. Adv Genet 82: 1‐45, 2013.
 124. Gootenberg JS , Abudayyeh OO , Lee JW , Essletzbichler P , Dy AJ , Joung J , Verdine V , Donghia N , Daringer NM , Freije CA , Myhrvold C , Bhattacharyya RP , Livny J , Regev A , Koonin E V. , Hung DT , Sabeti PC , Collins JJ , Zhang F . Nucleic acid detection with CRISPR‐Cas13a/C2c2. Science 356: 438‐442, 2017.
 125. Grabarek JB , Plusa B , Glover DM , Zernicka‐Goetz M . Efficient delivery of dsRNA into zona‐enclosed mouse oocytes and preimplantation embryos by electroporation. Genesis 32: 269‐276, 2002.
 126. Gratz SJ , Ukken FP , Rubinstein CD , Thiede G , Donohue LK , Cummings AM , O'Connor‐Giles KM . Highly specific and efficient CRISPR/Cas9‐catalyzed homology‐directed repair in Drosophila. Genetics 196: 961‐971, 2014.
 127. Grez M , Reichenbach J , Schwäble J , Seger R , Dinauer MC , Thrasher AJ . Gene therapy of chronic granulomatous disease: The engraftment dilemma. Mol Ther 19: 28‐35, 2011.
 128. Grissa I , Vergnaud G , Pourcel C . The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats. BMC Bioinformatics 8: 1‐10, 2007.
 129. Grissa I , Vergnaud G , Pourcel C . CRISPRFinder: A web tool to identify clustered regularly interspace short palindromic repeats. Nucleic Acids Res 35: 52‐57, 2007.
 130. Groner AC , Meylan S , Ciuffi A , Zangger N , Ambrosini G , Dénervaud N , Bucher P , Trono D . KRAB‐zinc finger proteins and KAP1 can mediate long‐range transcriptional repression through heterochromatin spreading. PLoS Genet 6: e1000869, 2010.
 131. Guilinger JP , Thompson DB , Liu DR . Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat Biotechnol 32: 577‐582, 2014.
 132. Gurumurthy CB , Takahashi G , Wada K , Miura H , Sato M , Ohtsuka M . GONAD: A novel CRISPR/Cas9 genome editing method that does not require ex vivo handling of embryos. Curr Protoc Hum Genet 88: 15.8.1‐15.8.12, 2016.
 133. Gutschner T , Haemmerle M , Genovese G , Draetta GF , Chin L . Post‐translational regulation of Cas9 during G1 enhances homology‐directed repair. Cell Rep 14: 1555‐1566, 2016.
 134. Hacein‐Bey‐Abina S , Le Deist F , Carlier F , Bouneaud C , Hue C , De Villartay JP , Thrasher AJ , Wulffraat N , Sorensen R , Dupuis‐Girod S , Fischer A , Davies EG , Kuis W , Leiva L , Cavazzana‐Calvo M . Sustained correction of X‐linked severe combined immunodeficiency by ex vivo gene therapy. N Engl J Med 346: 1185‐1193, 2002.
 135. Hacein‐Bey‐Abina S , Garrigue A , Wang GP , Soulier J , Lim A , Morillon E , Clappier E , Caccavelli L , Delabesse E , Beldjord K , Asnafi V , MacIntyre E , Dal Cortivo L , Radford I , Brousse N , Sigaux F , Moshous D , Hauer J , Borkhardt A , Belohradsky BH , Wintergerst U , Velez MC , Leiva L , Sorensen R , Wulffraat N , Blanche S , Bushman FD , Fischer A , Cavazzana‐Calvo M . Insertional oncogenesis in 4 patients after retrovirus‐mediated gene therapy of SCID‐X1. J Clin Invest 118: 3132‐3142, 2008.
 136. Hacein‐Bey‐Abina S , von Kalle C , Schmidt M , Le Deist F , Wulffraat N , McIntyre E , Radford I , Villeval JL , Fraser CC , Cavazzana‐Calvo M , Fischer A . A serious adverse event after successful gene therapy for X‐linked severe combined immunodeficiency. N Engl J Med 348: 255‐256, 2003.
 137. Hacein‐Bey‐Abina S , Von Kalle C , Schmidt M , McCormack MP , Wulffraat N , Leboulch P , Lim A , Osborne CS , Pawliuk R , Morillon E , Sorensen R , Forster A , Fraser P , Cohen JI , De Saint Basile G , Alexander I , Wintergerst U , Frebourg T , Aurias A , Stoppa‐Lyonnet D , Romana S , Radford‐Weiss I , Gross F , Valensi F , Delabesse E , Macintyre E , Sigaux F , Soulier J , Leiva LE , Wissler M , Prinz C , Rabbitts TH , Le Deist F , Fischer A , Cavazzana‐Calvo M . LMO2‐associated clonal T cell proliferation in two patients after gene therapy for SCID‐X1. Science 302: 415‐419, 2003.
 138. Hacein‐Bey H , Cavazzana‐Calvo M , Le Deist F , Dautry‐Varsat A , Hivroz C , Rivière I , Danos O , Heard JM , Sugamura K , Fischer A , De Saint Basile G . Gamma‐c gene transfer into SCID X1 patients’ B‐cell lines restores normal high‐affinity interleukin‐2 receptor expression and function. Blood 87: 3108‐3116, 1996.
 139. Halbert CL , Allen JM , Miller AD . Adeno‐associated virus type 6 (AAV6) vectors mediate efficient transduction of airway epithelial cells in mouse lungs compared to that of AAV2 vectors. J Virol 75: 6615‐6624, 2001.
 140. Hansen K , Coussens MJ , Sago J , Subramanian S , Gjoka M , Briner D . Genome editing with CompoZr custom zinc finger nucleases (ZFNs). J Vis Exp e3304, 2012.
 141. Harrington LB , Doxzen KW , Ma E , Liu JJ , Knott GJ , Edraki A , Garcia B , Amrani N , Chen JS , Cofsky JC , Kranzusch PJ , Sontheimer EJ , Davidson AR , Maxwell KL , Doudna JA . A broad‐spectrum inhibitor of CRISPR‐Cas9. Cell 170: 1224‐1233.e15, 2017.
 142. Harrington LB , Paez‐Espino D , Staahl BT , Chen JS , Ma E , Kyrpides NC , Doudna JA . A thermostable Cas9 with increased lifetime in human plasma. Nat Commun 8: 1424, 2017.
 143. He C , Gouble A , Bourdel A , Manchev V , Poirot L , Paques F , Duchateau P , Edelman A , Danos O . Lentiviral protein delivery of meganucleases in human cells mediates gene targeting and alleviates toxicity. Gene Ther 21: 759‐766, 2014.
 144. He X , Tan C , Wang F , Wang Y , Zhou R , Cui D , You W , Zhao H , Ren J , Feng B . Knock‐in of large reporter genes in human cells via CRISPR/Cas9‐induced homology‐dependent and independent DNA repair. Nucleic Acids Res 44: e85, 2016.
 145. Heigwer F , Kerr G , Boutros M . E‐CRISP: Fast CRISPR target site identification. Nat Methods 11: 122‐123, 2014.
 146. Hendel A , Bak RO , Clark JT , Kennedy AB , Ryan DE , Roy S , Steinfeld I , Lunstad BD , Kaiser RJ , Wilkens AB , Bacchetta R , Tsalenko A , Dellinger D , Bruhn L , Porteus MH . Chemically modified guide RNAs enhance CRISPR‐Cas genome editing in human primary cells. Nat Biotechnol 33: 985‐989, 2015.
 147. Hendel A , Kildebeck EJ , Fine EJ , Clark JT , Punjya N , Sebastiano V , Bao G , Porteus MH . Quantifying genome‐editing outcomes at endogenous loci with SMRT sequencing. Cell Rep 7: 293‐305, 2014.
 148. Henser‐Brownhill T , Monserrat J , Scaffidi P . Generation of an arrayed CRISPR‐Cas9 library targeting epigenetic regulators: From high‐content screens to in vivo assays. Epigenetics 2294: 1‐11, 2018.
 149. Hentze MW , Kulozik AE . A perfect message: RNA surveillance and nonsense‐mediated decay. Cell 96: 307‐310, 1999.
 150. Hess GT , Tycko J , Yao D , Bassik MC . Methods and applications of CRISPR‐mediated base editing in eukaryotic genomes. Mol Cell 68: 26‐43, 2017.
 151. Hilton IB , D'Ippolito AM , Vockley CM , Thakore PI , Crawford GE , Reddy TE , Gersbach CA . Epigenome editing by a CRISPR‐Cas9‐based acetyltransferase activates genes from promoters and enhancers. Nat Biotechnol 33: 510‐517, 2015.
 152. Hirano S , Nishimasu H , Ishitani R , Nureki O . Structural basis for the altered PAM specificities of engineered CRISPR‐Cas9. Mol Cell 61: 886‐894, 2016.
 153. Hirata R , Chamberlain J , Dong R , Russell DW . Targeted transgene insertion into human chromosomes by adeno‐associated virus vectors. Nat Biotechnol 20: 735‐738, 2002.
 154. Hoban MD , Lumaquin D , Kuo CY , Romero Z , Long J , Ho M , Young CS , Mojadidi M , Fitz‐Gibbon S , Cooper AR , Lill GR , Urbinati F , Campo‐Fernandez B , Bjurstrom CF , Pellegrini M , Hollis RP , Kohn DB . CRISPR/Cas9‐mediated correction of the sickle mutation in human CD34+ cells. Mol Ther 24: 1561‐1569, 2016.
 155. Hockemeyer D , Soldner F , Beard C , Gao Q , Mitalipova M , Dekelver RC , Katibah GE , Amora R , Boydston EA , Zeitler B , Meng X , Miller JC , Zhang L , Rebar EJ , Gregory PD , Urnov FD , Jaenisch R . Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc‐finger nucleases. 27: 851‐857, 2009.
 156. Hodgkins A , Farne A , Perera S , Grego T , Parry‐Smith DJ , Skarnes WC , Iyer V . WGE: A CRISPR database for genome engineering. Bioinformatics 31: 3078‐3080, 2015.
 157. Holkers M , Maggio I , Henriques SFD , Janssen JM , Cathomen T , Gonçalves MAFV . Adenoviral vector DNA for accurate genome editing with engineered nucleases. Nat Methods 11: 1051‐1057, 2014.
 158. Holmgaard A , Askou AL , Benckendorff JNE , Thomsen EA , Cai Y , Bek T , Mikkelsen JG , Corydon TJ . In vivo knockout of the Vegfa gene by lentiviral delivery of CRISPR/Cas9 in mouse retinal pigment epithelium cells. Mol Ther Nucleic Acids 9: 89‐99, 2017.
 159. Horii T , Arai Y , Yamazaki M , Morita S , Kimura M , Itoh M , Abe Y , Hatada I . Validation of microinjection methods for generating knockout mice by CRISPR/Cas‐mediated genome engineering. Sci Rep 4: 4513, 2014.
 160. Horlbeck MA , Witkowsky LB , Guglielmi B , Replogle JM , Gilbert LA , Villalta JE , Torigoe SE , Tjian R , Weissman JS . Nucleosomes impede Cas9 access to DNA in vivo and in vitro. Elife 5: pii: e12677, 2016.
 161. Hou Z , Zhang Y , Propson NE , Howden SE , Chu L , Sontheimer EJ , Thomson JA . Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis. Proc Natl Acad Sci USA 110: 15644‐15649, 2013.
 162. Howe SJ , Mansour MR , Schwarzwaelder K , Bartholomae C , Hubank M , Kempski H , Brugman MH , Pike‐Overzet K , Chatters SJ , De Ridder D , Gilmour KC , Adams S , Thornhill SI , Parsley KL , Staal FJT , Gale RE , Linch DC , Bayford J , Brown L , Quaye M , Kinnon C , Ancliff P , Webb DK , Schmidt M , Von Kalle C , Gaspar HB , Thrasher AJ . Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID‐X1 patients. J Clin Invest 118: 3143‐3150, 2008.
 163. Hsu PD , Lander ES , Zhang F . Development and applications of CRISPR‐Cas9 for genome engineering. Cell 157: 1262‐1278, 2014.
 164. Hsu PD , Scott DA , Weinstein JA , Ran FA , Konermann S , Agarwala V , Li Y , Fine EJ , Wu X , Shalem O , Cradick TJ , Marraffini LA , Bao G , Zhang F . DNA targeting specificity of RNA‐guided Cas9 nucleases. Nat Biotechnol 31: 827‐832, 2013.
 165. Hu JH , Miller SM , Geurts MH , Tang W , Chen L , Sun N , Zeina CM , Gao X , Rees HA , Lin Z , Liu DR . Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature 556: 57‐63, 2018.
 166. Hu Z , Ding W , Zhu D , Yu L , Jiang X , Wang X , Zhang C , Wang L , Ji T , Liu D , He D , Xia X , Zhu T , Wei J , Wu P , Wang C , Xi L , Gao Q , Chen G , Liu R , Li K , Li S , Wang S , Zhou J , Ma D , Wang H . TALEN‐mediated targeting of HPV oncogenes ameliorates HPV‐related cervical malignancy. J Clin Invest 125: 425‐436, 2015.
 167. Hu Z , Yu L , Zhu D , Ding W , Wang X , Zhang C , Wang L , Jiang X , Shen H , He D , Li K , Xi L , Ma D , Wang H . Disruption of HPV16‐E7 by CRISPR/Cas system induces apoptosis and growth inhibition in HPV16 positive human cervical cancer cells. Biomed Res Int 2014: 612823, 2014.
 168. Huang P , Xiao A , Zhou M , Zhu Z , Lin S , Zhang B . Heritable gene targeting in zebrafish using customized TALENs. Nat. Biotechnol. 29: 699‐700, 2011.
 169. Huang X , Wang JYJ , Lu X . Systems analysis of quantitative shRNA‐library screens identifies regulators of cell adhesion. BMC Syst Biol 2: 49, 2008.
 170. Huang Y , Paxton WA , Wolinsky SM , Neumann AU , Zhang L , He T , Kang S , Ceradini D , Jin Z , Yazdanbakhsh K , Kunstman K , Erickson D , Dragon E , Landau NR , Phair J , Ho DD , Koup RA . The role of a mutant CCR5 allele in HIV‐1 transmission and disease progression. Nat Med 2: 1240‐1243, 1996.
 171. Hur JK , Kim K , Been KW , Baek G , Ye S , Hur JW , Ryu SM , Lee YS , Kim JS . Targeted mutagenesis in mice by electroporation of Cpf1 ribonucleoproteins. Nat Biotechnol 34: 807‐808, 2016.
 172. Hütter G , Nowak D , Mossner M , Ganepola S , Müssig A , Allers K , Schneider T , Hofmann J , Kücherer C , Blau O , Blau IW , Hofmann WK , Thiel E . Long‐term control of HIV by CCR5 Delta32/Delta32 stem‐cell transplantation. N Engl J Med 360: 692‐698, 2009.
 173. Inoue N , Dong R , Hirata RK , Russell DW . Introduction of single base substitutions at homologous chromosomal sequences by adeno‐associated virus vectors. Mol Ther 3: 526‐530, 2001.
 174. Isaac RS , Jiang F , Doudna JA , Lim WA , Narlikar GJ , Almeida R . Nucleosome breathing and remodeling constrain CRISPR‐Cas9 function. Elife 5: pii: e13450, 2016.
 175. Ishino Y , Shinagawa H , Makino K , Amemura M , Nakata A . Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. J Bacteriol 169: 5429‐5433, 1987.
 176. Iwase H , Liu H , Wijkstrom M , Zhou H , Singh J , Hara H , Ezzelarab M , Long C , Klein E , Wagner R , Phelps C , Ayares D , Shapiro R , Humar A , Cooper DKC . Pig kidney graft survival in a baboon for 136 days: Longest life‐supporting organ graft survival to date. Xenotransplantation 22: 302‐309, 2015.
 177. Jacobi AM , Rettig GR , Turk R , Collingwood MA , Zeiner SA , Quadros RM , Harms DW , Bonthuis PJ , Gregg C , Ohtsuka M , Gurumurthy CB , Behlke MA . Simplified CRISPR tools for efficient genome editing and streamlined protocols for their delivery into mammalian cells and mouse zygotes. Methods 121‐122: 16‐28, 2017.
 178. Jacquier A , Dujon B . An intron‐encoded protein is active in a gene conversion process that spreads an intron into a mitochondrial gene. Cell 41: 383‐394, 1985.
 179. Jager L , Ehrhardt A . Persistence of high‐capacity adenoviral vectors as replication‐defective monomeric genomes in vitro and in murine liver. Hum Gene Ther 20: 883‐896, 2009.
 180. Jansen R , van Embden JDA , Gaastra W , Schouls LM . Identification of genes that are associated with DNA repeats in prokaryotes. Mol Microbiol 43: 1565‐1575, 2002.
 181. Jiang F , Doudna JA . CRISPR – Cas9 structures and mechanisms. AnnuRevBiophys 46: 505‐529, 2017.
 182. Jiang F , Zhou K , Ma L , Gressel S , Doudna JA . A Cas9‐guide RNA complex preorganized for target DNA recognition. Science 348: 1477‐1481, 2015.
 183. Jiang J , Jing Y , Cost GJ , Chiang JC , Kolpa HJ , Cotton AM , Carone DM , Carone BR , Shivak DA , Guschin DY , Pearl JR , Rebar EJ , Byron M , Gregory PD , Brown CJ , Urnov FD , Hall LL , Lawrence JB . Translating dosage compensation to trisomy 21. Nature 500: 296‐300, 2013.
 184. Jinek M , Chylinski K , Fonfara I , Hauer M , Doudna JA , Charpentier E . A programmable dual‐RNA‐guided DNA endonuclease in adaptive bacterial immunity. Science 337: 816‐822, 2012.
 185. Jinek M , East A , Cheng A , Lin S , Ma E , Doudna J . RNA‐programmed genome editing in human cells. Elife 2: e00471, 2013.
 186. Jinek M , Jiang F , Taylor DW , Sternberg SH , Kaya E , Ma E , Anders C , Hauer M , Zhou K , Lin S , Kaplan M , Iavarone AT , Charpentier E , Nogales E , Doudna JA . Structures of Cas9 endonucleases reveal RNA‐mediated conformational activation. Science 343: 1247997, 2014.
 187. Joglekar A V. , Hollis RP , Kuftinec G , Senadheera S , Chan R , Kohn DB . Integrase‐defective lentiviral vectors as a delivery platform for targeted modification of adenosine deaminase locus. Mol Ther 21: 1705‐1717, 2013.
 188. Johansen S , Embley TM , Willassen NP . A family of nuclear homing endonucleases. Nucleic Acids Res 21: 4405, 1993.
 189. Joshi R , Ho KK , Tenney K , Chen JH , Golden BL , Gimble FS . Evolution of I‐SceI Homing endonucleases with increased DNA recognition site specificity. J Mol Biol 405: 185‐200, 2011.
 190. Joung J , Engreitz JM , Konermann S , Abudayyeh OO , Verdine VK , Aguet F , Gootenberg JS , Sanjana NE , Wright JB , Fulco CP , Tseng YY , Yoon CH , Boehm JS , Lander ES , Zhang F . Genome‐scale activation screen identifies a lncRNA locus regulating a gene neighbourhood. Nature 548: 343‐346, 2017.
 191. Joung J , Konermann S , Gootenberg JS , Abudayyeh OO , Platt RJ , Brigham MD , Sanjana NE , Zhang F . Protocol: Genome‐scale CRISPR‐Cas9 knockout and transcriptional activation screening. bioRxiv 12: 059626, 2016.
 192. Jurica MS , Monnat Jr. RJ , Stoddard BL . DNA recognition and cleavage by the LAGLIDADG homing endonuclease I‐Cre I. Mol Cell 2: 469‐476, 1998.
 193. Kan Y , Ruis B , Takasugi T , Hendrickson EA . Mechanisms of precise genome editing using oligonucleotide donors. Genome Res 27: 1099‐1111, 2017.
 194. Kaneko T , Mashimo T . Simple genome editing of rodent intact embryos by electroporation. PLoS One 10: e0142755, 2015.
 195. Kaneko T , Sakuma T , Yamamoto T , Mashimo T . Simple knockout by electroporation of engineered endonucleases into intact rat embryos. Sci Rep 4, 2014.
 196. Kang HJ , Bartholomae CC , Paruzynski A , Arens A , Kim S , Yu SS , Hong Y , Joo CW , Yoon NK , Rhim JW , Kim JG , Von Kalle C , Schmidt M , Kim S , Ahn HS . Retroviral gene therapy for X‐linked Chronic Granulomatous Disease: Results from phase I/II trial. Mol Ther 19: 2092‐2101, 2011.
 197. Karvelis T , Gasiunas G , Miksys A , Barrangou R , Horvath P , Siksnys V . crRNA and tracrRNA guide Cas9‐mediated DNA interference in Streptococcus thermophilus. RNA Biol 10: 841‐851, 2013.
 198. Karvelis T , Gasiunas G , Siksnys V . Harnessing the natural diversity and in vitro evolution of Cas9 to expand the genome editing toolbox. Curr Opin Microbiol 37: 88‐94, 2017.
 199. Kaur K , Tandon H , Gupta AK , Kumar M . CrisprGE: A central hub of CRISPR/Cas‐based genome editing. Database (Oxford) 2015: bav055, 2015. doi: 10.1093/database/bav055.
 200. Kawano Y , Honda A . Gene targeting in rabbits: Single‐step generation of knock‐out rabbits by microinjection of CRISPR/Cas9 plasmids. Methods Mol Biol 1630: 109‐120, 2017.
 201. Kearns NA , Genga RMJ , Enuameh MS , Garber M , Wolfe SA , Maehr R . Cas9 effector‐mediated regulation of transcription and differentiation in human pluripotent stem cells. Development 141: 219‐223, 2014.
 202. Kelley ML , Strezoska Ž , He K , Vermeulen A , Smith A . Versatility of chemically synthesized guide RNAs for CRISPR‐Cas9 genome editing. J Biotechnol 233: 74‐83, 2016.
 203. Kelton WJ , Pesch T , Matile S , Reddy ST . Surveying the delivery methods of CRISPR/Cas9 for ex vivo mammalian cell engineering. Chim Int J Chem 70: 439‐442, 2016.
 204. Khan AO , Simms VA , Pike JA , Thomas SG , Morgan NV . CRISPR‐Cas9 mediated labelling allows for single molecule imaging and resolution. Sci Rep 7: 1‐9, 2017.
 205. Kiani S , Chavez A , Tuttle M , Hall RN , Chari R , Ter‐Ovanesyan D , Qian J , Pruitt BW , Beal J , Vora S , Buchthal J , Kowal EJ , Ebrahimkhani MR , Collins JJ , Weiss R , Church G . Cas9 gRNA engineering for genome editing, activation and repression. Nat Methods 12: 1051‐1054, 2015.
 206. Kim D , Bae S , Park J , Kim E , Kim S , Yu HR , Hwang J , Kim JI , Kim JS . Digenome‐seq: Genome‐wide profiling of CRISPR‐Cas9 off‐target effects in human cells. Nat Methods 12: 237‐243, 2015.
 207. Kim D , Kim J , Hur JK , Been KW , Yoon SH , Kim JS . Genome‐wide analysis reveals specificities of Cpf1 endonucleases in human cells. Nat Biotechnol 34: 863‐868, 2016.
 208. Kim E , Koo T , Park SW , Kim D , Kim K , Cho HY , Song DW , Lee KJ , Jung MH , Kim S , Kim JH , Kim JH , Kim JS . In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni. Nat Commun 8: 14500, 2017.
 209. Kim H , Um E , Cho SR , Jung C , Kim H , Kim JS . Surrogate reporters for enrichment of cells with nuclease‐induced mutations. Nat Methods 8: 941‐943, 2011.
 210. Kim S , Bae T , Hwang J , Kim JS . Rescue of high‐specificity Cas9 variants using sgRNAs with matched 5’ nucleotides. Genome Biol 18: 218, 2017.
 211. Kim S , Kim D , Cho SW , Kim J , Kim JS . Highly efficient RNA‐guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res 24: 1012‐1019, 2014.
 212. Kim Y , Cheong SA , Lee JG , Lee SW , Lee MS , Baek IJ , Sung YH . Generation of knockout mice by Cpf1‐mediated gene targeting. Nat Biotechnol 34: 808‐810, 2016.
 213. Kim YG , Cha J , Chandrasegaran S . Hybrid restriction enzymes: Zinc finger fusions to Fok I cleavage domain. Proc Natl Acad Sci U S A 93: 1156‐1160, 1996.
 214. Kim YG , Chandrasegaran S . Chimeric restriction endonuclease. Proc Natl Acad Sci 91: 883‐887, 1994.
 215. Kim YG , Durgesha M , Chandrasegaran S , Smith J . Chimeric restriction enzyme: Gal4 fusion to Fok cleavage domain. Biol Chem Hoppe Seyler 379: 489‐496, 1998.
 216. Kimura J , Nguyen ST , Liu H , Taira N , Miki Y , Yoshida K . A functional genome‐wide RNAi screen identifies TAF1 as a regulator for apoptosis in response to genotoxic stress. Nucleic Acids Res 36: 5250‐5259, 2008.
 217. Klann TS , Black JB , Chellappan M , Safi A , Song L , Hilton IB , Crawford GE , Reddy TE , Gersbach CA . CRISPR–Cas9 epigenome editing enables high‐throughput screening for functional regulatory elements in the human genome. Nat Biotechnol 35: 561‐568, 2017.
 218. Kleinstiver BP , Pattanayak V , Prew MS , Tsai SQ , Nguyen NT , Zheng Z , Joung JK . High‐fidelity CRISPR‐Cas9 nucleases with no detectable genome‐wide off‐target effects. Nature 529: 490‐495, 2016.
 219. Kleinstiver BP , Prew MS , Tsai SQ , Topkar VV , Nguyen NT , Zheng Z , Gonzales APW , Li Z , Peterson RT , Yeh JRJ , Aryee MJ , Joung JK . Engineered CRISPR‐Cas9 nucleases with altered PAM specificities. Nature 523: 481‐485, 2015.
 220. Kohli M , Rago C , Lengauer C , Kinzler KW , Vogelstein B . Facile methods for generating human somatic cell gene knockouts using recombinant adeno‐associated viruses. Nucleic Acids Res 32: e3, 2004.
 221. Kohn DB . Gene therapy for genetic haematological disorders and immunodeficiencies. J Intern Med 249(4): 379‐390, 2001.
 222. Koike‐Yusa H , Li Y , Tan EP , Velasco‐Herrera MDC , Yusa K . Genome‐wide recessive genetic screening in mammalian cells with a lentiviral CRISPR‐guide RNA library. Nat Biotechnol 32: 267‐273, 2014.
 223. Komor AC , Kim YB , Packer MS , Zuris JA , Liu DR . Programmable editing of a target base in genomic DNA without double‐stranded DNA cleavage. Nature 533: 420‐424, 2016.
 224. Konermann S , Brigham MD , Trevino AE , Joung J , Abudayyeh OO , Barcena C , Hsu PD , Habib N , Gootenberg JS , Nishimasu H , Nureki O , Zhang F . Genome‐scale transcriptional activation by an engineered CRISPR‐Cas9 complex. Nature 517: 583‐588, 2014.
 225. Koo T , Lee J , Kim J‐S . Measuring and Reducing Off‐Target Activities of Programmable Nucleases Including CRISPR‐Cas9. Mol Cells 38: 475‐481, 2015.
 226. Koonin EV , Makarova KS , Zhang F . Diversity, classification and evolution of CRISPR‐Cas systems. Curr Opin Microbiol 37: 67‐78, 2017.
 227. Korkmaz G , Lopes R , Ugalde AP , Nevedomskaya E , Han R , Myacheva K , Zwart W , Elkon R , Agami R . Functional genetic screens for enhancer elements in the human genome using CRISPR‐Cas9. Nat Biotechnol 34: 192‐198, 2016.
 228. Kotin RM , Siniscalco M , Samulski RJ , Zhu XD , Hunter L , Laughlin CA , McLaughlin S , Muzyczka N , Rocchi M , Berns KI . Site‐specific integration by adeno‐associated virus. Proc Natl Acad Sci U S A 87: 2211‐2215, 1990.
 229. Krejci L , Altmannova V , Spirek M , Zhao X . Homologous recombination and its regulation. Nucleic Acids Res. 40: 5795‐5818, 2012.
 230. Kulcsár PI , Tálas A , Huszár K , Ligeti Z , Tóth E , Weinhardt N , Fodor E , Welker E . Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage. Genome Biol 18: 1‐17, 2017.
 231. Kumar M , Keller B , Makalou N , Sutton RE . Systematic determination of the packaging limit of lentiviral vectors. Hum Gene Ther 12: 1893‐1905, 2001.
 232. Labun K , Montague TG , Gagnon JA , Thyme SB , Valen E . CHOPCHOP v2: A web tool for the next generation of CRISPR genome engineering. Nucleic Acids Res 44: W272–W276, 2016.
 233. Lambowitz AM , Belfort M . Introns as mobile genetic elements. Annu Rev Biochem 62: 587‐622, 1993.
 234. De Lange O , Binder A , Lahaye T . From dead leaf, to new life: TAL effectors as tools for synthetic biology. Plant J 78: 753‐771, 2014.
 235. Lanphier E , Urnov F , Haecker SE , Werner M , Smolenski J . Don't edit the human germ line. Nature 519: 410‐411, 2015.
 236. Lee CS , Bishop ES , Zhang R , Yu X , Farina EM , Yan S , Zhao C , Zeng Z , Shu Y , Wu X , Lei J , Li Y , Zhang W , Yang C , Wu K , Wu Y , Ho S , Athiviraham A , Lee MJ , Wolf JM , Reid RR , He TC . Adenovirus‐mediated gene delivery: Potential applications for gene and cell‐based therapies in the new era of personalized medicine. Genes Dis 4: 43‐63, 2017.
 237. Lensing S V. , Marsico G , Hänsel‐Hertsch R , Lam EY , Tannahill D , Balasubramanian S. DSBCapture: In situ capture and sequencing of DNA breaks. Nat Methods 13: 855‐857, 2016.
 238. LeProust EM , Peck BJ , Spirin K , McCuen HB , Moore B , Namsaraev E , Caruthers MH . Synthesis of high‐quality libraries of long (150mer) oligonucleotides by a novel depurination controlled process. Nucleic Acids Res 38: 2522‐2540, 2010.
 239. Li C , Guan X , Du T , Jin W , Wu B , Liu Y , Wang P , Hu B , Griffin GE , Shattock RJ , Hu Q . Inhibition of HIV‐1 infection of primary CD4+ T‐cells by gene editing of CCR5 using adenovirus‐delivered CRISPR/Cas9. J Gen Virol 96: 2381‐2393, 2015.
 240. Li C , Qi R , Singleterry R , Hyle J , Balch A , Li X , Sublett J , Berns H , Valentine M , Valentine V , Sherr CJ . Simultaneous Gene Editing by Injection of mRNAs Encoding Transcription Activator‐Like Effector Nucleases into Mouse Zygotes. Mol Cell Biol 34: 1649‐1658, 2014.
 241. Li G , Zhang X , Zhong C , Mo J , Quan R , Yang J , Liu D , Li Z , Yang H , Wu Z . Small molecules enhance CRISPR/Cas9‐mediated homology‐directed genome editing in primary cells. Sci Rep 7: 8943, 2017.
 242. Li H , Beckman KA , Pessino V , Huang B , Weissman JS , Leonetti MD . Design and specificity of long ssDNA donors for CRISPR‐based knock‐in. bioRxiv (2017). doi: 10.1101/178905.
 243. Li H , Haurigot V , Doyon Y , Li T , Wong SY , Bhagwat AS , Malani N , Anguela XM , Sharma R , Ivanciu L , Murphy SL , Finn JD , Khazi FR , Zhou S , Paschon DE , Rebar EJ , Bushman FD , Gregory PD , Holmes MC , High KA . In vivo genome editing restores haemostasis in a mouse model of haemophilia. Nature 475: 217‐221, 2011.
 244. Li K , Wang G , Andersen T , Zhou P , Pu WT . Optimization of genome engineering approaches with the CRISPR/Cas9 system. PLoS One 9: e105779, 2014.
 245. Li P , Kleinstiver BP , Leon MY , Prew MS , Navarro‐Gomez D , Greenwald SH , Pierce EA , Joung JK , Liu Q . Allele‐specific CRISPR/Cas9 genome editing of the single‐base P23H mutation for rhodopsin associated dominant retinitis pigmentosa. Cris J 1: 55‐64, 2018.
 246. Liang P , Xu Y , Zhang X , Ding C , Huang R , Zhang Z , Lv J , Xie X , Chen Y , Li Y , Sun Y , Bai Y , Songyang Z , Ma W , Zhou C , Huang J . CRISPR/Cas9‐mediated gene editing in human tripronuclear zygotes. Protein Cell 6: 363‐372, 2015.
 247. Liao HK , Hatanaka F , Araoka T , Reddy P , Wu MZ , Sui Y , Yamauchi T , Sakurai M , O'Keefe DD , Núñez‐Delicado E , Guillen P , Campistol JM , Wu CJ , Lu LF , Esteban CR , Izpisua Belmonte JC . In vivo target gene activation via CRISPR/Cas9‐mediated trans‐epigenetic modulation. Cell 17: 1495‐1507, 2017. doi: 10.1016/j.cell.2017.10.025.
 248. Lieber MR , Ma Y , Pannicke U , Schwarz K . Mechanism and regulation of human non‐homologous DNA end‐joining. Nat Rev Mol Cell Biol 4: 712‐720, 2003.
 249. Liesche C , Venkatraman L , Aschenbrenner S , Grosse S , Grimm D , Eils R , Beaudouin J . Death receptor‐based enrichment of Cas9‐expressing cells. BMC Biotechnol 16: 17, 2016.
 250. Limberis MP , Vandenberghe LH , Zhang L , Pickles RJ , Wilson JM . Transduction efficiencies of novel AAV vectors in mouse airway epithelium in vivo and human ciliated airway epithelium in vitro. Mol Ther 17: 294‐301, 2009.
 251. Lin S , Staahl BT , Alla RK , Doudna JA . Enhanced homology‐directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. Elife 3: e04766, 2014.
 252. Lisowski L , Tay SS , Alexander IE . Adeno‐associated virus serotypes for gene therapeutics. Curr Opin Pharmacol 24: 59‐67, 2015.
 253. Liu H , Wei Z , Dominguez A , Li Y , Wang X , Qi LS . CRISPR‐ERA: A comprehensive design tool for CRISPR‐mediated gene editing, repression and activation. Bioinformatics 31: 3676‐3678, 2015.
 254. Liu J , Gaj T , Wallen MC , Barbas CF . Improved cell‐penetrating zinc‐finger nuclease proteins for precision genome engineering. Mol Ther Nucleic Acids 4: e232, 2015.
 255. Liu Q , Xia Z , Case CC . Validated zinc finger protein designs for all 16 GNN DNA triplet targets. J Biol Chem 277: 3850‐3856, 2002.
 256. Liu XS , Wu H , Ji X , Stelzer Y , Wu X , Czauderna S , Shu J , Dadon D , Young RA , Jaenisch R . Editing DNA methylation in the mammalian genome. Cell 167: 233‐247.e17, 2016.
 257. Llorente B , Smith CE , Symington LS . Break‐induced replication: What is it and what is it for? Cell Cycle 7: 859‐864, 2008.
 258. Lloy A , Vickery ON , Laugel B . Beyond the antigen receptor: Editing the genome of T‐cells for cancer adoptive cellular therapies. Front Immunol 4: 221, 2013.
 259. Lombardo A , Genovese P , Beausejour CM , Colleoni S , Lee YLL , Kim KA , Ando D , Urnov FD , Galli C , Gregory PD , Holmes MC , Naldini L . Gene editing in human stem cells using zinc finger nucleases and integrase‐defective lentiviral vector delivery. Nat Biotechnol 25: 1298‐1306, 2007.
 260. Luo J , Emanuele MJ , Li D , Creighton CJ , Schlabach MR , Westbrook TF , Wong KK , Elledge SJ . A genome‐wide RNAi screen identifies multiple synthetic lethal interactions with the Ras oncogene. Cell 137: 835‐848, 2009.
 261. Ma AC , McNulty MS , Poshusta TL , Campbell JM , Martínez‐Gálvez G , Argue DP , Lee HB , Urban MD , Bullard CE , Blackburn PR , Man TK , Clark KJ , Ekker SC . FusX: A rapid one‐step transcription activator‐like effector assembly system for genome Science. Hum Gene Ther 27: 451‐463, 2016.
 262. Ma H , Marti‐Gutierrez N , Park SW , Wu J , Lee Y , Suzuki K , Koski A , Ji D , Hayama T , Ahmed R , Darby H , Van Dyken C , Li Y , Kang E , Park AR , Kim D , Kim S , Gong J , Gu Y , Xu X , Battaglia D , Krieg SA , Lee DM , Wu DH , Wolf DP , Heitner SB , Belmonte JCI , Amato P , Kim JS , Kaul S , Mitalipov S . Correction of a pathogenic gene mutation in human embryos. Nature 548: 413‐419, 2017.
 263. Maeder ML , Linder SJ , Cascio VM , Fu Y , Ho QH , Joung JK . CRISPR RNA‐guided activation of endogenous human genes. Nat Methods 10: 977‐979, 2014.
 264. Maeder ML , Thibodeau‐Beganny S , Osiak A , Wright DA , Anthony RM , Eichtinger M , Jiang T , Foley JE , Winfrey RJ , Townsend JA , Unger‐Wallace E , Sander JD , Müller‐Lerch F , Fu F , Pearlberg J , Göbel C , Dassie JPP , Pruett‐Miller SM , Porteus MH , Sgroi DC , Iafrate AJ , Dobbs D , McCray PB , Cathomen T , Voytas DF , Joung JK . Rapid “open‐source” engineering of customized zinc‐finger nucleases for highly efficient gene modification. Mol Cell 31: 294‐301, 2008.
 265. Maggio I , Holkers M , Liu J , Janssen JM , Chen X , Gonçalves MAFV . Adenoviral vector delivery of RNA‐guided CRISPR/Cas9 nuclease complexes induces targeted mutagenesis in a diverse array of human cells. Sci Rep 4: 5105, 2015.
 266. Makarova KS , Haft DH , Barrangou R , Brouns SJJ , Charpentier E , Horvath P , Moineau S , Mojica FJM , Wolf YI , Yakunin AF , Van Der Oost J , Koonin EV . Evolution and classification of the CRISPR‐Cas systems. Nat Rev Microbiol 9: 467‐477, 2011.
 267. Mali P , Aach J , Stranges PB , Esvelt KM , Kosuri S , Yang L , Church GM . Cas9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol 31: 1‐15, 2013.
 268. Mali P , Yang L , Esvelt KM , Aach J , Guell M , DiCarlo JE , Norville JE , Church GM . RNA‐guided human genome engineering via Cas9. Science 339: 823‐826, 2013.
 269. Malkova A , Ira G . Break‐induced replication: Functions and molecular mechanism. Curr Opin Genet Dev 23: 271‐279, 2013.
 270. Mandell JG , Barbas CF . Zinc Finger Tools: Custom DNA‐binding domains for transcription factors and nucleases. Nucleic Acids Res 34: W516‐W523, 2006.
 271. Manno CS , Chew AJ , Hutchison S , Larson PJ , Herzog RW , Arruda VR , Tai SJ , Ragni M V. , Thompson A , Ozelo M , Couto LB , Leonard DGB , Johnson FA , McClelland A , Scallan C , Skarsgard E , Flake AW , Kay MA , High KA , Glader B . AAV‐mediated factor IX gene transfer to skeletal muscle in patients with severe hemophilia B. Blood 101: 2963‐2972, 2003.
 272. Mao Z , Bozzella M , Seluanov A , Gorbunova V . DNA repair by nonhomologous end joining and homologous recombination during cell cycle in human cells. Cell Cycle 7: 2902‐2906, 2008.
 273. Marmor M , Sheppard HW , Donnell D , Bozeman S , Celum C , Buchbinder S , Koblin B , Seage GR . Homozygous and heterozygous CCR5‐Delta32 genotypes are associated with resistance to HIV infection. J Acquir Immune Defic Syndr 27: 472‐481, 2001.
 274. Marraffini LA , Sontheimer EJ. CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science 322: 1843‐1845, 2008.
 275. Martinson JJ , Chapman NH , Rees DC , Liu YT , Clegg JB . Global distribution of the CCR5 gene 32‐basepair deletion. Nat Genet 16: 100‐103, 1997.
 276. Mashal RD , Koontz J , Sklar J . Detection of mutations by cleavage of DNA heteroduplexes with bacteriophage resolvases. Nat Genet 9: 177‐183, 1995.
 277. Mashiko D , Fujihara Y , Satouh Y , Miyata H , Isotani A , Ikawa M . Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA. Sci Rep 3: 3355, 2013.
 278. McGinn J , Marraffini LA . Molecular mechanisms of CRISPR‐Cas spacer acquisition. Nat Rev Microbiol 17: 7‐12, 2018. doi: https://doi.org/10.1038/s41579‐018‐0071‐7.
 279. Mehta A , Haber JE . Sources of DNA double‐strand breaks and models of recombinational DNA repair. Cold Spring Harb Perspect Biol 6: 1‐19, 2014.
 280. Meng X , Noyes MB , Zhu LJ , Lawson ND , Wolfe A . Targeted gene inactivation in zebrafish using engineered zinc finger nucleases. Nat Biotechnol 26: 695‐701, 2008.
 281. Merkert S , Martin U . Targeted genome engineering using designer nucleases: State of the art and practical guidance for application in human pluripotent stem cells. Stem Cell Res 16: 377‐386, 2016.
 282. Metzakopian E , Strong A , Iyer V , Hodgkins A , Tzelepis K , Antunes L , Friedrich MJ , Kang Q , Davidson T , Lamberth J , Hoffmann C , Davis GD , Vassiliou GS , Skarnes WC , Bradley A . Enhancing the genome editing toolbox: Genome wide CRISPR arrayed libraries. Sci Rep 7: 1‐9, 2017.
 283. Miller JC , Holmes MC , Wang J , Guschin DY , Lee YL , Rupniewski I , Beausejour CM , Waite AJ , Wang NS , Kim KA , Gregory PD , Pabo CO , Rebar EJ . An improved zinc‐finger nuclease architecture for highly specific genome editing. Nat Biotechnol 25: 778‐785, 2007.
 284. Miller JC , Tan S , Qiao G , Barlow KA , Wang J , Xia DF , Meng X , Paschon DE , Leung E , Hinkley SJ , Dulay GP , Hua KL , Ankoudinova I , Cost GJ , Urnov FD , Zhang HS , Holmes MC , Zhang L , Gregory PD , Rebar EJ . A TALE nuclease architecture for efficient genome editing. Nat Biotechnol 29: 143‐150, 2011.
 285. Milner PF , Charache S . Life span of carbamylated red cells in sickle cell anemia. J Clin Invest 52: 3161‐3171, 1973.
 286. Mobley JL . Is rheumatoid arthritis a consequence of natural selection for enhanced tuberculosis resistance?> Med Hypotheses 62: 839‐843, 2004.
 287. Moehle EA , Rock JM , Lee YL , Jouvenot Y , Dekelver RC , Gregory PD , Urnov FD , Holmes MC . Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases. Proc Natl Acad Sci 104: 3055‐3060, 2007.
 288. Mojica FJM , Díez‐Villaseñor C , Soria E , Juez G . Biological significance of a family of regularly spaced repeats in the genomes of Archaea, Bacteria and mitochondria. Mol Microbiol 36: 244‐246, 2000.
 289. Montague TG , Cruz JM , Gagnon JA , Church GM , Valen E . CHOPCHOP: A CRISPR/Cas9 and TALEN web tool for genome editing. Nucleic Acids Res 42: W401‐W407, 2014.
 290. Morita S , Noguchi H , Horii T , Nakabayashi K , Kimura M , Okamura K , Sakai A , Nakashima H , Hata K , Nakashima K , Hatada I . Targeted DNA demethylation in vivo using dCas9–peptide repeat and scFv–TET1 catalytic domain fusions. Nat Biotechnol 34: 1060‐1065, 2016.
 291. Morton J , Davis MW , Jorgensen EM , Carroll D . Induction and repair of zinc‐finger nuclease‐targeted double‐strand breaks in Caenorhabditis elegans somatic cells. Proc Natl Acad Sci 103: 16370‐16375, 2006.
 292. Moscou MJ , Bogdanove AJ . A simple cipher governs DNA recognition by TAL effectors. Science 326: 1501, 2009.
 293. Moure CM , Gimble FS , Quiocho FA . The crystal structure of the gene targeting homing endonuclease I‐SceI reveals the origins of its target site specificity. J Mol Biol 334: 685‐695, 2003.
 294. Mullenders J , Fabius AWM , Madiredjo M , Bernards R , Beijersbergen RL . A large scale shRNA barcode screen identifies the circadian clock component ARNTL as putative regulator of the p53 tumor suppressor pathway. PLoS One 4: e4798, 2009.
 295. Nagaoka‐Yasuda R , Matsuo N , Perkins B , Limbaeck‐Stokin K , Mayford M . An RNAi‐based genetic screen for oxidative stress resistance reveals retinol saturase as a mediator of stress resistance. Free Radic Biol Med 43: 781‐788, 2007.
 296. Naito Y , Hino K , Bono H , Ui‐Tei K . CRISPRdirect: Software for designing CRISPR/Cas guide RNA with reduced off‐target sites. Bioinformatics 31: 1120‐1123, 2015.
 297. Naso MF , Tomkowicz B , Perry WL , Strohl WR . Adeno‐associated virus (AAV) as a vector for gene therapy. BioDrugs 31: 317‐334, 2017.
 298. Nishimasu H , Ran FA , Hsu PD , Konermann S , Shehata SI , Dohmae N , Ishitani R , Zhang F , Nureki O . Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156: 935‐949, 2014.
 299. Niu D , Wei HJ , Lin L , George H , Wang T , Lee I‐H , Zhao HY , Wang Y , Kan Y , Shrock E , Lesha E , Wang G , Luo Y , Qing Y , Jiao D , Zhao H , Zhou X , Wang S , Wei H , Güell M , Church GM , Yang L . Inactivation of porcine endogenous retrovirus in pigs using CRISPR‐Cas9. Science 357: 1303‐1307, 2017. doi: 10.1126/science.aan4187.
 300. Noda T , Oji A , Ikawa M . Genome editing in mouse zygotes and embryonic stem cells by introducing SgRNA/Cas9 expressing plasmids. Methods Mol Biol 1630: 67‐80, 2017.
 301. Noguchi M , Yi H , Rosenblatt HM , Filipovich AH , Adelstein S , Modi WS , McBride OW , Leonard WJ . Interleukin‐2 receptor γ chain mutation results in X‐linked severe combined immunodeficiency in humans. Cell 73: 147‐157, 1993.
 302. O'Brien A , Bailey TL . GT‐Scan: Identifying unique genomic targets. Bioinformatics 30: 2673‐2675, 2014.
 303. Orlando SJ , Santiago Y , DeKelver RC , Freyvert Y , Boydston EA , Moehle EA , Choi VM , Gopalan SM , Lou JF , Li J , Miller JC , Holmes MC , Gregory PD , Urnov FD , Cost GJ . Zinc‐finger nuclease‐driven targeted integration into mammalian genomes using donors with limited chromosomal homology. Nucleic Acids Res 38: e152, 2010.
 304. Ormond KE , Mortlock DP , Scholes DT , Bombard Y , Brody LC , Faucett WA , Garrison NA , Hercher L , Isasi R , Middleton A , Musunuru K , Shriner D , Virani A , Young CE . Human germline genome editing. Am J Hum Genet 101: 167‐176, 2017.
 305. Paludan SR . Activation and regulation of DNA‐driven immune responses. Microbiol Mol Biol Rev 79: 225‐241, 2015.
 306. Parant JM , George SA , Pryor R , Wittwer CT , Yost HJ . A rapid and efficient method of genotyping zebrafish mutants. Dev Dyn 238: 3168‐3174, 2009.
 307. Park J , Bae S , Kim JS . Cas‐Designer: A web‐based tool for choice of CRISPR‐Cas9 target sites. Bioinformatics 31: 4014‐4016, 2015.
 308. Park KE , Kaucher AV , Powell A , Waqas MS , Sandmaier SES , Oatley MJ , Park CH , Tibary A , Donovan DM , Blomberg LA , Lillico SG , Whitelaw CBA , Mileham A , Telugu BP , Oatley JM . Generation of germline ablated male pigs by CRISPR/Cas9 editing of the NANOS2 gene. Sci Rep 7: 2017.
 309. Pavletich N , Pabo C. Zinc finger‐DNA recognition: Crystal structure of a Zif268‐DNA complex at 2.1 A. Science 252: 809‐817, 1991.
 310. Perez‐Pinera P , Kocak DD , Vockley CM , Adler AF , Kabadi M , Polstein LR , Thakore PI , Glass KA , David G , Leong KW , Guilak F , Crawford GE , Timothy E , Gersbach CA . RNA‐guided gene activation by CRISPR‐Cas9‐based transcription factors. Nat Methods 10: 973‐976, 2013.
 311. Perez EE , Wang J , Miller JC , Jouvenot Y , Kim KA , Liu O , Wang N , Lee G , Bartsevich VV , Lee YL , Guschin DY , Rupniewski I , Waite AJ , Carpenito C , Carroll RG , Orange JS , Urnov FD , Rebar EJ , Ando D , Gregory PD , Riley JL , Holmes MC , June CH . Establishment of HIV‐1 resistance in CD4+ T cells by genome editing using zinc‐finger nucleases. Nat Biotechnol 26: 808‐816, 2008.
 312. Pierce AJ , Johnson RD , Thompson LH , Jasin M . XRCC3 promotes homology‐directed repair of DNA damage in mammalian cells. Genes Dev 13: 2633‐2638, 1999.
 313. Plessis A , Perrin A , Haber JE , Dujon B . Site‐specific recombination determined by I‐SceI, a mitochondrial group I intron‐encoded endonuclease expressed in the yeast nucleus. Genetics 130: 451‐460, 1992.
 314. Pliatsika V , Rigoutsos I. “Off‐Spotter”: Very fast and exhaustive enumeration of genomic lookalikes for designing CRISPR/Cas guide RNAs. Biol Direct 10, 2015.
 315. Popp MW , Maquat LE . Leveraging rules of nonsense‐mediated mRNA decay for genome engineering and personalized medicine. Cell 165: 1319‐1332, 2016.
 316. Porteus MH , Baltimore D . Chimeric nucleases stimulate gene targeting in human cells. Science 300: 763, 2003.
 317. Pruett‐Miller SM , Davis GD . Donor plasmid design for codon and single base genome editing using zinc finger nucleases. Methods Mol Biol 1239: 219‐229, 2015.
 318. Pruett‐Miller SM , Reading DW , Porter SN , Porteus MH . Attenuation of zinc finger nuclease toxicity by small‐molecule regulation of protein levels. PLoS Genet 5: e1000376, 2009.
 319. Prykhozhij SV , Rajan V , Gaston D , Berman JN . CRISPR MultiTargeter: A web tool to find common and unique CRISPR single guide RNA targets in a set of similar sequences. PLoS One 10: e0119372, 2015.
 320. Pulido‐Quetglas C , Aparicio‐Prat E , Arnan C , Polidori T , Hermoso T , Palumbo E , Ponomarenko J , Guigo R , Johnson R . Scalable design of paired CRISPR guide RNAs for genomic deletion. PLoS Comput Biol 13: 1‐18, 2017.
 321. Qi LS , Larson MH , Gilbert LA , Doudna JA , Weissman JS , Arkin AP , Lim WA . Repurposing CRISPR as an RNA‐guided platform for sequence‐specific control of gene expression. Cell 152: 1173‐1183, 2013.
 322. Qi Y , Zhang Y , Zhang F , Baller JA , Cleland SC , Ryu Y , Starker CG , Voytas DF . Increasing frequencies of site‐specific mutagenesis and gene targeting in Arabidopsis by manipulating DNA repair pathways. Genome Res 23: 547‐554, 2013.
 323. Qin W , Dion SL , Kutny PM , Zhang Y , Cheng AW , Jillette NL , Malhotra A , Geurts AM , Chen YG , Wang H . Efficient CRISPR/cas9‐mediated genome editing in mice by zygote electroporation of nuclease. Genetics 200: 423‐430, 2015.
 324. Qin W , Kutny PM , Maser RS , Dion SL , Lamont JD , Zhang Y , Perry GA , Wang H . Generating mouse models using CRISPR‐Cas9‐mediated genome editing. Curr Protoc Mouse Biol 6: 39‐66, 2016.
 325. Quadros RM , Miura H , Harms DW , Akatsuka H , Sato T , Aida T , Redder R , Richardson GP , Inagaki Y , Sakai D , Buckley SM , Seshacharyulu P , Batra SK , Behlke MA , Zeiner SA , Jacobi AM , Izu Y , Thoreson WB , Urness LD , Mansour SL , Ohtsuka M , Gurumurthy CB . Easi‐CRISPR: A robust method for one‐step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins. Genome Biol 18: 92, 2017.
 326. Quan L , Chen X , Liu A , Zhang Y , Guo X , Yan S , Liu Y . PD‐1 blockade can restore functions of T‐cells in Epstein‐Barr virus‐positive diffuse large B‐cell lymphoma in vitro. PLoS One 10: e0136476, 2015.
 327. Rahdar M , McMahon MA , Prakash TP , Swayze EE , Bennett CF , Cleveland DW . Synthetic CRISPR RNA‐Cas9–guided genome editing in human cells. Proc Natl Acad Sci 112(51): E7110‐E7117, 2015. doi: 10.1073/pnas.1520883112.
 328. Ramirez CL , Foley JE , Wright DA , Müller‐Lerch F , Rahman SH , Cornu TI , Winfrey RJ , Sander JD , Fu F , Townsend JA , Cathomen T , Voytas DF , Joung JK . Unexpected failure rates for modular assembly of engineered zinc fingers. Nat Methods 5: 374‐375, 2008.
 329. Ran FA , Cong L , Yan WX , Scott DA , Gootenberg JS , Kriz AJ , Zetsche B , Shalem O , Wu X , Makarova KS , Koonin EV , Sharp PA , Zhang F . In vivo genome editing using Staphylococcus aureus Cas9. Nature 520: 186‐191, 2015.
 330. Ran FA , Hsu PD , Lin CY , Gootenberg JS , Konermann S , Trevino AE , Scott DA , Inoue A , Matoba S , Zhang Y , Zhang F . Double nicking by RNA‐guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154: 1380‐1389, 2013.
 331. Rauch BJ , Silvis MR , Hultquist JF , Waters CS , McGregor MJ , Krogan NJ , Bondy‐Denomy J . Inhibition of CRISPR‐Cas9 with bacteriophage proteins. Cell 168: 150‐158.e10, 2017.
 332. Regalado A . Who owns the biggest biotech discovery of the century [Online]. MIT Technol Rev 2014. https://www.technologyreview.com/s/532796/who‐owns‐the‐biggest‐biotech‐discovery‐of‐the‐century/ [accessed May 10, 2018].
 333. Reyon D , Tsai SQ , Khgayter C , Foden JA , Sander JD , Joung JK . FLASH assembly of TALENs for high‐throughput genome editing. Nat Biotechnol 30: 460‐465, 2012.
 334. Rinaldi FC , Doyle LA , Stoddard BL , Bogdanove AJ . The effect of increasing numbers of repeats on TAL effector DNA binding specificity. Nucleic Acids Res 45: 6960‐6970, 2017.
 335. Ringo A . Understanding deafness: not everyone wants to be “fixed” [Online]. Atl.: 1‐7, 2013. http://www.theatlantic.com/health/archive/2013/08/understanding‐deafness‐not‐everyone‐wants‐to‐be‐fixed/278527/ [accessed May 6, 2018].
 336. Rosenbluh J , Xu H , Harrington W , Gill S , Wang X , Vazquez F , Root DE , Tsherniak A , Hahn WC . Complementary information derived from CRISPR Cas9 mediated gene deletion and suppression. Nat Commun 8: 1‐8, 2017.
 337. Rothkamm K , Kruger I , Thompson LH , Lobrich M . Pathways of DNA double‐strand break repair during the mammalian cell cycle. Mol Cell Biol 23: 5706‐5715, 2003.
 338. Rouet P , Smih F , Jasin M . Introduction of double‐strand breaks into the genome of mouse cells by expression of a rare‐cutting endonuclease. Mol Cell Biol 14: 8096‐8106, 1994.
 339. Rouet P , Smih F , Jasin M . Expression of a site‐specific endonuclease stimulates homologous recombination in mammalian cells. Proc Natl Acad Sci U S A 91: 6064‐6068, 1994.
 340. Sabeti PC , Walsh E , Schaffner SF , Varilly P , Fry B , Hutcheson HB , Cullen M , Mikkelsen TS , Roy J , Patterson N , Cooper R , Reich D , Altshuler D , O'Brien S , Lander ES . The case for selection at CCR5‐Delta32. PLoS Biol 3: e378, 2005.
 341. Sakane Y , Suzuki K ich T , Yamamoto T . A simple protocol for loss‐of‐function analysis in Xenopus tropicalis founders using the CRISPR‐Cas system. Methods Mol Biol 1630: 189‐203, 2017.
 342. Samulski RJ , Muzyczka N . AAV‐mediated gene therapy for research and therapeutic purposes. Annu Rev Virol 1: 427‐451, 2014.
 343. Sander JD , Cade L , Khayter C , Reyon D , Peterson RT , Joung JK , Yeh JRJ . Targeted gene disruption in somatic zebrafish cells using engineered TALENs. Nat. Biotechnol. 29: 697‐698, 2011.
 344. Sander JD , Joung JK . CRISPR‐Cas systems for editing, regulating and targeting genomes. Nat Biotechnol 32: 347‐350, 2014.
 345.Sangamo. The most precise genome editing technology [Online]. 2018. https://www.sangamo.com/technology/genome-editing [accessed April 12, 2018].
 346. Sankaran VG , Menne TF , Xu J , Akie TE , Lettre G , Van Handel B , Mikkola HKA , Hirschhorn JN , Cantor AB , Orkin SH . Human fetal hemoglobin expression is regulated by the developmental stage‐specific repressor BCL11A. Science 322: 1839‐1842, 2008.
 347. Santiago Y , Chan E , Liu PQ , Orlando S , Zhang L , Urnov FD , Holmes MC , Guschin D , Waite A , Miller JC , Rebar EJ , Gregory PD , Klug A , Collingwood TN . Targeted gene knockout in mammalian cells by using engineered zinc‐finger nucleases. Proc Natl Acad Sci U S A 105: 5809‐5814, 2008.
 348. Sapranauskas R , Gasiunas G , Fremaux C , Barrangou R , Horvath P , Siksnys V . The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli. Nucleic Acids Res 39: 9275‐9282, 2011.
 349. Scaringe SA . RNA oligonucleotide synthesis via 5′‐silyl‐ 2′‐orthoester chemistry. Methods 23: 206‐217, 2001.
 350. Schiroli G , Ferrari S , Conway A , Jacob A , Capo V , Albano L , Plati T , Castiello MC , Sanvito F , Gennery AR , Bovolenta C , Palchaudhuri R , Scadden DT , Holmes MC , Villa A , Sitia G , Lombardo A , Genovese P , Naldini L . Preclinical modeling highlights the therapeutic potential of hematopoietic stem cell gene editing for correction of SCID‐X1. Sci Transl Med 9, 2017.
 351. Schmidt T , Schmid‐Burgk JL , Hornung V . Synthesis of an arrayed sgRNA library targeting the human genome. Sci Rep 5: 1‐10, 2015.
 352. Schreiber T , Bonas U . Repeat 1 of TAL effectors affects target specificity for the base at position zero. Nucleic Acids Res 42: 7160‐7169, 2014.
 353. Schwarzwaelder K , Howe SJ , Schmidt M , Brugman MH , Deichmann A , Glimm H , Schmidt S , Prinz C , Wissler M , King DJS , Zhang F , Parsley KL , Gilmour KC , Sinclair J , Bayford J , Peraj R , Pike‐Overzet K , Staal FJT , De Ridder D , Kinnon C , Abel U , Wagemaker G , Gaspar HB , Thrasher AJ , Von Kalle C . Gammaretrovirus‐mediated correction of SCID‐X1 is associated with skewed vector integration site distribution in vivo. J Clin Invest 117: 2241‐2249, 2007.
 354. Segal DJ , Beerli RR , Blancafort P , Dreier B , Effertz K , Huber A , Koksch B , Lund CV , Magnenat L , Valente D , Barbas CF . Evaluation of a modular strategy for the construction of novel polydactyl zinc finger DNA‐binding proteins. Biochemistry 42: 2137‐2148, 2003.
 355. Seligman LM , Chisholm KM , Chevalier BS , Chadsey MS , Edwards ST , Savage JH , Veillet AL . Mutations altering the cleavage specificity of a homing endonuclease. Nucleic Acids Res 30: 3870‐3879, 2002.
 356. Sentmanat MF , Peters ST , Florian CP , Connelly JP , Pruett‐Miller SM . A Survey of validation strategies for CRISPR‐Cas9 editing. Sci Rep 8: 1‐8, 2018.
 357. Shalem O , Sanjana NE , Hartenian E , Shi X , Scott DA , Heckl D , Ebert BL , Root DE , Doench JG . Genome‐scale CRISPR‐Cas9 knockout screening in human cells. Science 343: 84‐87, 2014.
 358. Sharma P , Allison JP . Immune checkpoint targeting in cancer therapy: Toward combination strategies with curative potential. Cell 161: 205‐214, 2015.
 359. Sharma R , Anguela XM , Doyon Y , Wechsler T , DeKelver RC , Sproul S , Paschon DE , Miller JC , Davidson RJ , Shivak D , Zhou S , Rieders J , Gregory PD , Holmes MC , Rebar EJ , High KA . In vivo genome editing of the albumin locus as a platform for protein replacement therapy. Blood 126: 1777‐1784, 2015.
 360. Shemin D , Rittenberg D . The life span of the human red blood cell. J Biol Chem 166: 627‐636, 1946.
 361. Shi J , Wang E , Milazzo JP , Wang Z , Kinney JB , Vakoc CR . Discovery of cancer drug targets by CRISPR‐Cas9 screening of protein domains. Nat Biotechnol 33: 661‐667, 2015.
 362. Shibata A . Regulation of repair pathway choice at two‐ended DNA double‐strand breaks. Mutat Res 803‐805: 51‐55, 2017.
 363. Shin J , Jiang F , Liu JJ , Bray NL , Rauch BJ , Baik SH , Nogales E , Bondy‐Denomy J , Corn JE , Doudna JA . Disabling Cas9 by an anti‐CRISPR DNA mimic. Sci Adv 3: e1701620, 2017.
 364. Shmakov S , Abudayyeh OO , Makarova KS , Wolf YI , Gootenberg JS , Semenova E , Minakhin L , Joung J , Konermann S , Severinov K , Zhang F , Koonin E V . Discovery and functional characterization of diverse class 2 CRISPR‐Cas systems. Mol Cell 60: 385‐397, 2015.
 365. Silva GH , Belfort M , Wende W , Pingoud A . From monomeric to homodimeric endonucleases and back: Engineering novel specificity of LAGLIDADG enzymes. J Mol Biol 361: 744‐754, 2006.
 366. Singh R , Kuscu C , Quinlan A , Qi Y , Adli M . Cas9‐chromatin binding information enables more accurate CRISPR off‐target prediction. Nucleic Acids Res 43, 2015.
 367. Slaymaker IM , Gao L , Zetsche B , Scott DA , Yan WX , Zhang F . Rationally engineered Cas9 nucleases with improved specificity. Science 351: 84‐88, 2016.
 368. Smih F , Rouet P , Romanienko PJ , Jasin M . Double‐strand breaks at the target locus stimulate gene targeting in embryonic stem cells. Nucleic Acids Res 23: 5012‐5019, 1995.
 369. Smith C , Gore A , Yan W , Abalde‐Atristain L , Li Z , He C , Wang Y , Brodsky RA , Zhang K , Cheng L , Ye Z . Whole‐genome sequencing analysis reveals high specificity of CRISPR/Cas9 and TALEN‐based genome editing in human iPSCs. Cell Stem Cell 15: 12‐13, 2014.
 370. Smith I , Greenside PG , Natoli T , Lahr DL , Wadden D , Tirosh I , Narayan R , Root DE , Golub TR , Subramanian A , Doench JG . Evaluation of RNAi and CRISPR technologies by large‐scale gene expression profiling in the Connectivity Map. PLoS Biol 15: 1‐23, 2017.
 371. Smith J , Grizot S , Arnould S , Duclert A , Epinat JC , Chames P , Prieto J , Redondo P , Blanco FJ , Bravo J , Montoya G , Pâques F , Duchateau P . A combinatorial approach to create artificial homing endonucleases cleaving chosen sequences. Nucleic Acids Res 34: e149, 2006.
 372. Soldner F , Laganière J , Cheng AW , Hockemeyer D , Gao Q , Alagappan R , Khurana V , Golbe LI , Myers RH , Lindquist S , Zhang L , Guschin D , Fong LK , Vu BJ , Meng X , Urnov FD , Rebar EJ , Gregory PD , Zhang HS , Jaenisch R . Generation of isogenic pluripotent stem cells differing exclusively at two early onset parkinson point mutations. Cell 146: 318‐331, 2011.
 373. Song J , Yang D , Xu J , Zhu T , Chen YE , Zhang J . RS‐1 enhances CRISPR/Cas9‐ and TALEN‐mediated knock‐in efficiency. Nat Commun 7: 1‐7, 2016.
 374. Soudais C , Shiho T , Sharara LI , Guy‐Grand D , Taniguchi T , Fischer A , Di Santo JP . Stable and functional lymphoid reconstitution of common cytokine receptor gamma chain deficient mice by retroviral‐mediated gene transfer. Blood 95: 3071‐3077, 2000.
 375. Stemmer M , Thumberger T , Del Sol Keyer M , Wittbrodt J , Mateo JL . CCTop: An intuitive, flexible and reliable CRISPR/Cas9 target prediction tool. PLoS One 10: e0124633, 2015.
 376. Sternberg SH , LaFrance B , Kaplan M , Doudna JA . Conformational control of DNA target cleavage by CRISPR‐Cas9. Nature 527: 110‐113, 2015.
 377. Sternberg SH , Redding S , Jinek M , Greene EC , Doudna JA . DNA interrogation by the CRISPR RNA‐guided endonuclease Cas9. Nature 507: 62‐67, 2014.
 378. Stoddard BL . Homing endonuclease structure and function. Q. Rev. Biophys. 38: 49‐95, 2005.
 379. Straub A , Lahaye T. Zinc fingers, TAL effectors, or Cas9‐based DNA binding proteins: What's best for targeting desired genome loci? Mol Plant 6: 1384‐1387, 2013.
 380. Streubel J , Blücher C , Landgraf A , Boch J . TAL effector RVD specificities and efficiencies. Nat Biotechnol. 30: 593‐595, 2012.
 381. Strezoska Ž , Perkett MR , Chou ET , Maksimova E , Anderson EM , McClelland S , Kelley ML , Vermeulen A , Smith A . High‐content analysis screening for cell cycle regulators using arrayed synthetic crRNA libraries. J Biotechnol 251: 189‐200, 2017.
 382. Su S , Hu B , Shao J , Shen B , Du J , Du Y , Zhou J , Yu L , Zhang L , Chen F , Sha H , Cheng L , Meng F , Zou Z , Huang X , Liu B . CRISPR‐Cas9 mediated efficient PD‐1 disruption on human primary T cells from cancer patients. Sci Rep 6: P53, 2016.
 383. Surosky RT , Urabe M , Godwin SG , McQuiston SA , Kurtzman GJ , Ozawa K , Natsoulis G . Adeno‐associated virus Rep proteins target DNA sequences to a unique locus in the human genome. [Online]. J Virol 71: 7951‐7959, 1997. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=192153&tool=pmcentrez&rendertype=abstract [accessed March 23, 2018].
 384. Sussman D , Chadsey M , Fauce S , Engel A , Bruett A , Monnat R , Stoddard BL , Seligman LM . Isolation and characterization of new homing endonuclease specificities at individual target site positions. J Mol Biol 342: 31‐41, 2004.
 385. Swarup S , Yang Y , Kingsley MT , Gabriel DW . An Xanthomonas citri pathogenicity gene, pthA, pleiotropically encodes gratuitous avirulence on nonhosts. Mol plant‐microbe Interact. 5: 204‐213, 1992.
 386. Szczepek M , Brondani V , Buchel J , Serrano L , Segal DJ , Cathomen T . Structure‐based redesign of the dimerization interface reduces the toxicity of zinc‐finger nucleases. Nat Biotechnol 25: 786‐793, 2007.
 387. Takeuchi R , Choi M , Stoddard BL . Redesign of extensive protein‐DNA interfaces of meganucleases using iterative cycles of in vitro compartmentalization. Proc Natl Acad Sci 111: 4061‐4066, 2014.
 388. Takeuchi R , Choi M , Stoddard BL . Engineering of customized meganucleases via in vitro compartmentalization and in cellulo optimization. In: Chromosomal Mutagenesis, 2nd Ed. New York: Humana Press, 2014, pp. 105‐132.
 389. Tan J , Martin SE . Validation of synthetic CRISPR reagents as a tool for arrayed functional genomic screening. PLoS One 11: 1‐14, 2016.
 390. Tanenbaum ME , Gilbert LA , Qi LS , Weissman JS , Vale RD . A protein‐tagging system for signal amplification in gene expression and fluorescence imaging. Cell 159: 635‐646, 2014.
 391. Taylor J , Woodcock S . A perspective on the future of high‐throughput RNAi screening: Will CRISPR cut out the competition or can RNAi help guide the way? J Biomol Screen 20: 1040‐1051, 2015.
 392. Terns MP , Terns RM . CRISPR‐based adaptive immune systems. Curr Opin Microbiol 14: 321‐327, 2011.
 393. Thakore PI , Song L , Safi A , Shivakumar K , Kabadi AM , Reddy TE , Crawford GE , Gersbach CA . Highly specific epigenome editing by CRISPR/Cas9 repressors for silencing of distal regulatory elements. Nat Methods 12: 1143‐1149, 2015.
 394. Thomas KR , Folger KR , Capecchi MR . High frequency targeting of genes to specific sites in the mammalian genome. Cell 44: 419‐428, 1986.
 395. Thompson AJ , Yuan X , Kudlicki W , Herrin DL . Cleavage and recognition pattern of a double‐strand‐specific endonuclease (I‐CreI) encoded by the chloroplast 23S rRNA intron of Chlamydomonas reinhardtii. Gene 119: 247‐251, 1992.
 396. Thyme SB , Boissel SJS , Arshiya Quadri S , Nolan T , Baker DA , Park RU , Kusak L , Ashworth J , Baker D . Reprogramming homing endonuclease specificity through computational design and directed evolution. Nucleic Acids Res 42: 2564‐2576, 2014.
 397. Traxler EA , Yao Y , Wang YD , Woodard KJ , Kurita R , Nakamura Y , Hughes JR , Hardison RC , Blobel GA , Li C , Weiss MJ . A genome‐editing strategy to treat β‐hemoglobinopathies that recapitulates a mutation associated with a benign genetic condition. Nat Med 22: 987‐990, 2016.
 398. Tsai SQ . Discovering the genome‐wide activity of CRISPR‐Cas nucleases. ACS Chem Biol 13: 305‐308, 2018.
 399. Tsai SQ , Nguyen NT , Malagon‐Lopez J , Topkar VV , Aryee MJ , Joung JK . CIRCLE‐seq: A highly sensitive in vitro screen for genome‐wide CRISPR‐Cas9 nuclease off‐targets. Nat Methods 14: 607‐614, 2017.
 400. Tsai SQ , Wyvekens N , Khayter C , Foden JA , Thapar V , Reyon D , Goodwin MJ , Aryee MJ , Joung K . Dimeric CRISPR RNA‐guided FokI nucleases for highly specific genome editing. Nat Biotechnol 32: 569‐576, 2014.
 401. Tsai SQ , Zheng Z , Nguyen NT , Liebers M , Topkar VV , Thapar V , Wyvekens N , Khayter C , Iafrate AJ , Le LP , Aryee MJ , Joung JK . GUIDE‐seq enables genome‐wide profiling of off‐target cleavage by CRISPR‐Cas nucleases. Nat Biotechnol 33: 187‐198, 2015.
 402. Tschöp K , Conery AR , Litovchick L , DeCaprio JA , Settleman J , Harlow E , Dyson N . A kinase shRNA screen links LATS2 and the pRB tumor suppressor. Genes Dev 25: 814‐830, 2011.
 403. Tsubota T , Sezutsu H . Genome editing of silkworms. In: Methods in Molecular Biology, edited by Hatada I. New York: Humana Press, 2017, pp. 205‐218.
 404. Urnov FD , Miller JC , Lee YLL , Beausejour CM , Rock JM , Augustus S , Jamieson AC , Porteus MH , Gregory PD , Holmes MC . Highly efficient endogenous human gene correction using designed zinc‐finger nucleases. Nature 435: 646‐651, 2005.
 405. Valton J , Daboussi F , Leduc S , Molina R , Redondo P , Macmaster R , Montoya G , Duchateau P . 5′‐cytosine‐phosphoguanine (CpG) methylation impacts the activity of natural and engineered meganucleases. J Biol Chem 287: 30139‐30150, 2012.
 406. Vanamee ÉS , Santagata S , Aggarwal AK . FokI requires two specific DNA sites for cleavage. J Mol Biol 309: 69‐78, 2001.
 407. Varga CM , Tedford NC , Thomas M , Klibanov AM , Griffith LG , Lauffenburger DA . Quantitative comparison of polyethylenimine formulations and adenoviral vectors in terms of intracellular gene delivery processes. Gene Ther 12: 1023‐1032, 2005.
 408. Veres A , Gosis BS , Ding Q , Collins R , Ragavendran A , Brand H , Erdin S , Talkowski ME , Musunuru K . Low incidence of off‐target mutations in individual CRISPR‐Cas9 and TALEN targeted human stem cell clones detected by whole‐genome sequencing. Cell Stem Cell 15: 27‐30, 2014.
 409. Vojta A , Dobrinic P , Tadic V , Bockor L , Korac P , Julg B , Klasic M , Zoldos V . Repurposing the CRISPR‐Cas9 system for targeted DNA methylation. Nucleic Acids Res 44: 5615‐5628, 2016.
 410. Vora S , Tuttle M , Cheng J , Church G . Next stop for the CRISPR revolution: RNA‐guided epigenetic regulators. FEBS J 283: 3181‐3193, 2016.
 411. Wang B , Li K , Wang A , Reiser M , Saunders T , Lockey RF , Wang JW . Highly efficient CRISPR/HDR‐mediated knock‐in for mouse embryonic stem cells and zygotes. Biotechniques 59: 201‐208, 2015.
 412. Wang GP , Berry CC , Malani N , Leboulch P , Fischer A , Hacein‐Bey‐Abina S , Cavazzana‐Calvo M , Bushman FD . Dynamics of gene‐modified progenitor cells analyzed by tracking retroviral integration sites in a human SCID‐X1 gene therapy trial. Blood 115: 4356‐4366, 2010.
 413. Wang H , Yang H , Shivalila CS , Dawlaty MM , Cheng AW , Zhang F , Jaenisch R . One‐step generation of mice carrying mutations in multiple genes by CRISPR/cas‐mediated genome engineering. Cell 153: 910‐918, 2013.
 414. Wang T , Wei JJ , Sabatini DM , Lander ES . Genetic screens in human cells using the CRISPR‐Cas9 system. Science 343: 80‐85, 2014.
 415. Wang X , Wang Y , Wu X , Wang J , Wang Y , Qiu Z , Chang T , Huang H , Lin RJ , Yee JK . Unbiased detection of off‐target cleavage by CRISPR‐Cas9 and TALENs using integrase‐defective lentiviral vectors. Nat Biotechnol 33: 175‐179, 2015.
 416. Warner E . Reviewing CRISPR, the scientific discovery of the century [Online]. Labiotech.eu 2016. https://labiotech.eu/crispr‐review‐patent‐war/ [accessed May 10, 2018].
 417. Wefers B , Bashir S , Rossius J , Wurst W , Kühn R . Gene editing in mouse zygotes using the CRISPR/Cas9 system. Methods 121‐122: 55‐67, 2017.
 418. Wilmut I , Schnieke AE , McWhir J , Kind AJ , Campbell KHS . Viable offspring derived from fetal and adult mammalian cells. Nature 385: 810‐813, 1997.
 419. Wong N , Liu W , Wang X . WU‐CRISPR: Characteristics of functional guide RNAs for the CRISPR/Cas9 system. Genome Biol 16 2015.
 420. Wright AV , Sternberg SH , Taylor DW , Staahl BT , Bardales JA , Kornfeld JE , Doudna JA . Rational design of a split‐Cas9 enzyme complex. Proc Natl Acad Sci 112: 2984‐2989, 2015.
 421. Wu X , Scott DA , Kriz AJ , Chiu AC , Hsu PD , Dadon DB , Cheng AW , Trevino AE , Konermann S , Chen S , Jaenisch R , Zhang F , Sharp PA . Genome‐wide binding of the CRISPR endonuclease Cas9 in mammalian cells. Nat Biotechnol 32: 670‐676, 2014.
 422. Xie S , Shen B , Zhang C , Huang X , Zhang Y . sgRNAcas9: A software package for designing CRISPR sgRNA and evaluating potential off‐target cleavage sites. PLoS One 9: e100448, 2014.
 423. Xiong T , Meister GE , Workman RE , Kato NC , Spellberg MJ , Turker F , Timp W , Ostermeier M , Novina CD . Targeted DNA methylation in human cells using engineered dCas9‐methyltransferases. Sci Rep 7: 1‐14, 2017.
 424. Xu J , Peng C , Sankaran VG , Shao Z , Esrick EB , Chong BG , Ippolito GC , Fujiwara Y , Ebert BL , Tucker PW , Orkin SH . Correction of sickle cell disease in adult mice by interference with fetal hemoglobin silencing. Science 334: 993‐996, 2011.
 425. Xu L , Park KH , Zhao L , Xu J , El Refaey M , Gao Y , Zhu H , Ma J , Han R . CRISPR‐mediated genome editing restores dystrophin expression and function in mdx mice. Mol Ther 24: 564‐569, 2016.
 426. Xu X , Tao Y , Gao X , Zhang L , Li X , Zou W , Ruan K , Wang F , Xu GL , Hu R . A CRISPR‐based approach for targeted DNA demethylation. Cell Discov 2, 2016.
 427. Yamamoto Y , Bliss J , Gerbi SA . Whole organism genome editing: Targeted large DNA insertion via ObLiGaRe nonhomologous end‐joining in vivo capture. G3(Bethesda) 5: 1843‐1847, 2015.
 428. Yan WX , Mirzazadeh R , Garnerone S , Scott D , Schneider MW , Kallas T , Custodio J , Wernersson E , Li Y , Gao L , Federova Y , Zetsche B , Zhang F , Bienko M , Crosetto N . BLISS is a versatile and quantitative method for genome‐wide profiling of DNA double‐strand breaks. Nat Commun 8: 1‐9, 2017.
 429. Yang H , Wang H , Shivalila CS , Cheng AW , Shi L , Jaenisch R . One‐step generation of mice carrying reporter and conditional alleles by CRISPR/Cas mediated genome engineering. Cell 154: 1370‐1379, 2013.
 430. Yang J , Zhang Y , Yuan P , Zhou Y , Cai C , Ren Q , Wen D , Chu C , Qi H , Wei W . Complete decoding of TAL effectors for DNA recognition. Cell Res. 24: 628‐631, 2014.
 431. Yang Z , Steentoft C , Hauge C , Hansen L , Thomsen AL , Niola F , Vester‐Christensen MB , Frödin M , Clausen H , Wandall HH , Bennett EP . Fast and sensitive detection of indels induced by precise gene targeting. Nucleic Acids Res 43: e59, 2015.
 432. Yeung ML , Houzet L , Yedavalli VSRK , Jeang KT . A genome‐wide short hairpin RNA screening of jurkat T‐cells for human proteins contributing to productive HIV‐1 replication. J Biol Chem 284: 19463‐19473, 2009.
 433. Yin H , Song CQ , Dorkin JR , Zhu LJ , Li Y , Wu Q , Park A , Yang J , Suresh S , Bizhanova A , Gupta A , Bolukbasi MF , Walsh S , Bogorad RL , Gao G , Weng Z , Dong Y , Koteliansky V , Wolfe SA , Langer R , Xue W , Anderson DG . Therapeutic genome editing by combined viral and non‐viral delivery of CRISPR system components in vivo. Nat Biotechnol 34: 328‐333, 2016.
 434. Yin H , Song CQ , Suresh S , Wu Q , Walsh S , Rhym LH , Mintzer E , Bolukbasi MF , Zhu LJ , Kauffman K , Mou H , Oberholzer A , Ding J , Kwan SY , Bogorad RL , Zatsepin T , Koteliansky V , Wolfe SA , Xue W , Langer R , Anderson DG . Structure‐guided chemical modification of guide RNA enables potent non‐viral in vivo genome editing. Nat Biotechnol 35: 1179‐1187, 2017.
 435. Yu C , Liu Y , Ma T , Liu K , Xu S , Zhang Y , Liu H , La Russa M , Xie M , Sheng D , Qi LS . Small molecules enhance CRISPR genome editing in pluripotent stem cells. Cell Stem Cell 16: 142‐147, 2015.
 436. Zaiss AK , Muruve DA . Immune responses to adeno‐associated virus vectors. Curr Gene Ther 5: 323‐331, 2005.
 437. Zalatan JG , Lee ME , Almeida R , Gilbert LA , Whitehead EH , La Russa M , Tsai JC , Weissman JS , Dueber JE , Qi LS , Lim WA . Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds. Cell 160: 339‐350, 2015.
 438. Zetsche B , Gootenberg JS , Abudayyeh OO , Slaymaker IM , Makarova KS , Essletzbichler P , Volz SE , Joung J , Van Der Oost J , Regev A , Koonin EV , Zhang F . Cpf1 is a single RNA‐guided endonuclease of a class 2 CRISPR‐Cas system. Cell 163: 759‐771, 2015.
 439. Zetsche B , Heidenreich M , Mohanraju P , Fedorova I , Kneppers J , Degennaro EM , Winblad N , Choudhury SR , Abudayyeh OO , Gootenberg JS , Wu WY , Scott DA , Severinov K , Van Der Oost J , Zhang F . Multiplex gene editing by CRISPR‐Cpf1 using a single crRNA array. Nat Biotechnol 35: 31‐34, 2017.
 440. Zhang H , McCarty N . CRISPR‐Cas9 technology and its application in haematological disorders. Br J Haematol 175: 208‐225, 2016.
 441. Zhang Y , Long C , Li H , McAnally JR , Baskin KK , Shelton JM , Bassel‐Duby R , Olson EN . CRISPR‐Cpf1 correction of muscular dystrophy mutations in human cardiomyocytes and mice. Sci Adv 3: 2017.
 442. Zheng T , Hou Y , Zhang P , Zhang Z , Xu Y , Zhang L , Niu L , Yang Y , Liang D , Yi F , Peng W , Feng W , Yang Y , Chen J , Zhu YY , Zhang LH , Du Q . Profiling single‐guide RNA specificity reveals a mismatch sensitive core sequence. Sci Rep 7: 40638, 2017.
 443. Zhou M , Hu Z , Qiu L , Zhou T , Feng M , Hu Q , Zeng B , Li Z , Sun Q , Wu Y , Liu X , Wu L , Liang D . Seamless genetic conversion of SMN2 to SMN1 via CRISPR/Cpf1 and single‐stranded oligodeoxynucleotides in spinal muscular atrophy patient‐specific iPSCs. Hum Gene Ther 29: 1252‐1263, 2018. doi: 10.1089/hum.2017.255.
 444. Zhou Y , Zhu S , Cai C , Yuan P , Li C , Huang Y , Wei W . High‐throughput screening of a CRISPR/Cas9 library for functional genomics in human cells. Nature 509: 487‐491, 2014.
 445. Zhu LJ , Holmes BR , Aronin N , Brodsky MH . CRISPRseek: A Bioconductor package to identify target‐specific guide RNAs for CRISPR‐Cas9 genome‐editing systems. PLoS One 9: e108424, 2014.
 446. Zou J , Maeder ML , Mali P , Pruett‐Miller SM , Thibodeau‐Beganny S , Chou BK , Chen G , Ye Z , Park IH , Daley GQ , Porteus MH , Joung JK , Cheng L . Gene targeting of a disease‐related gene in human induced pluripotent stem and embryonic stem cells. Cell Stem Cell 5: 97‐110, 2009.

 

Teaching Material

S. N. Porter, R. M. Levine, S. M. Pruett-Miller. A Practical Guide to Genome Editing Using Targeted Nuclease Technologies. Compr Physiol 9: 2019, 665-714.

Didactic Synopsis

Major Teaching Points:

  • Targeted nucleases make sequence-specific breaks in DNA. The sequence changes induced by repair of these breaks is genome editing.
  • DNA double-strand breaks stimulate DNA repair pathways including nonhomologous end joining (NHEJ) and homology directed repair (HDR). NHEJ can be used to disrupt a gene via insertions or deletions of DNA bases at the break site. HDR requires a homologous DNA template and can be used to replace or introduce new DNA sequences at the break site.
  • Scientists have discovered and developed increasingly user-friendly tools for genome editing. These targeted nucleases include meganucleases, zinc-finger nucleases (ZFNs), TAL effector nucleases (TALENs), and CRISPR–Cas.
  • CRISPR–Cas, the most recently developed of these targeted nucleases has become very popular because it can be efficiently programmed to target a DNA sequence of interest using a RNA molecule that can complementary base pair with the target.
  • The CRISPR–Cas system has also been adapted to make changes to DNA without double-strand breaks. These technologies include base editors and epigenetic modifiers.
  • CRISPR–Cas can be used to create custom cell lines and animal models to aid basic and translational research.
  • Genome editing holds great therapeutic potential for human diseases, and there are current human clinical trials testing various nuclease-based therapies.
  • Editing the human genome, especially germline cells or embryos, raises many ethical questions. There is much debate about how genome editing should be governed and regulated.

Didactic Legends

The figures—in a freely downloadable PowerPoint format—can be found on the Images tab along with the formal legends published in the article. The following legends to the same figures are written to be useful for teaching.

Figure 1 Trends in publication of genome engineering technologies. This figure demonstrates the massive growth of the gene editing field over the last 50 years by quantifying publications and highlights the impact made by the introduction of each targeted nuclease platform. Red arrows indicate year of first publication of gene editing in mammalian cells with the indicated nuclease technology.

Figure 2 Homodimer and monomeric meganucleases bound to their DNA target sites. Teaching points: Meganucleases’ DNA binding and DNA cutting activities cannot be separated. For homodimer meganucleases (A), two proteins must bind a roughly palindromic DNA sequences close together to allow interaction and creation of a DNA double strand break between the two binding sites. For monomer meganucleses (B), a single protein is composed of two domains that bind different DNA sequences fused by a protein linker. As with (A), when both of these domains bind to DNA, a break is created between the two sites.

Figure 3 Pair of zinc finger nucleases (ZFNs) bound to their DNA target sites. This figure shows two 4 finger ZFNs bound to DNA. Each zinc finger (ZF) contacts approximately 3 base pairs of DNA and each ZFN half-site is ∼12 base pairs. DNA breaks are created in the spacer region by dimerization of the non-specific nuclease FokI.

Figure 4 Pair of transcription activator-like effector nucleases (TALENs) bound to their DNA target sites. Key points: TALENs bind in the opposite orientation from ZFNs. This figure shows two 17.5- repeat TALENs bound to genomic DNA. The repeat amino acid sequence is shown, with the repeat variable di-residues (RVD) highlighted in red. These two residues are responsible for the DNA base specificity of that repeat. DNA breaks are created in the spacer region by dimerization of the non-specific nuclease FokI.

Figure 5 CRISPR Immune Response Pathway. CRISPR-Cas systems developed in bacteria as an immune system against viral infection. The first time a virus infects a bacterium, short DNA sequences (protospacers) are integrated into the bacterial genome within the CRISPR array as spacers. These spacers are transcribed and processed into mature crRNA, waiting to recognize complimentary DNA. If this occurs, the RNA-Cas complex is guided by the crRNA to specifically bind and cleave the foreign DNA, causing a double stranded break that destroys the DNA and prevents infection.

Figure 6 CRISPR–Cas9 CRISPR RNA (crRNA) and single-guide RNA (sgRNA) bound to their DNA target sites. Key points: CRISPR–Cas9 nucleases are comprised of a Cas9 protein (blue) bound to a guide RNA. This guide can either be (A) a sequence-specific crRNA (purple) and a general tracrRNA (red) or (B) a combined single gRNA (sgRNA). The DNA target site has 20 nucleotides that are complementary to the first 20 nucleotides of the guide RNA. The PAM site (orange) is found on the nontarget genomic DNA strand immediately after the target site.

Figure 7 Comparison of Cas proteins with distinct mechanisms. This figure shows three Cas nuclease proteins that have different mechanisms of nucleotide cleavage. (A) Cas9 requires a PAM sequence of NGG and creates a blunt DNA double-strand break. (B) The gRNA for Cas12a is much shorter than for Cas9 and creates a staggered DNA double-strand break resulting in 5 nucleotide DNA overhangs or “sticky ends.” The PAM sequence for Cas12a is thymidine rich. (C) Cas13a binds to mRNA cleaves the mRNA outside of the target site. Cas13a binds a target sequence directly after a A, U or C base.

Figure 8 Double-Strand Break Repair Pathways Used for Genome Editing. This figure details the two main repair pathways used by the cell to repair double-strand breaks (DSBs), non-homologous end joining (NHEJ) and homology-directed repair (HDR). NHEJ, the preferred repair pathway, is designed to repair DSBs with precise end joining, however targeted nuclease activity can introduce insertions or deletions at the cleavage site, resulting in gene disruptions. Two DSBs at nearby sites can induce large deletions or inversions, and DSBs on two different chromosomes can lead to translocations. HDR naturally repairs DSBs using a sister chromatid as a repair template. Donor templates can be introduced into cells to introduce precise changes into the targeted DNA.

Figure 9 On-target assays for indel quantification. This figure shows commonly used assays for detecting targeted cleavage and subsequent repair via nonhomologous end-joining (NHEJ). Teaching points: most on-target assays are either PCR-based (A, B, C and F) or require expression of a reporter gene (D and E) in order to identify successful genomic edits.

Figure 10 On-Target assays for confirming desired editing events. This figure shows commonly used assays for detecting targeted cleavage and subsequent repair by homology directed repair (HDR). Teaching points: most on-target assays are either PCR-based (A and B) or require gene expression (C and D) in order to identify successful HDR events.

Figure 11 On-target assays for indel quantification and confirming desired editing events (knockins and knockouts). This figure shows commonly used assays for detecting targeted cleavage and subsequent repair by both NHEJ and HDR. Teaching points: Both assays are PCR based and sequencing based, which precludes larger HDR events from being detected using either assay. For larger inserts, assays shown in Figure 10B, C, and D are more suitable.

Figure 12 Modifications to the CRISPR-Cas9 platform to reduce off-target editing. This figure shows guide RNAs that have been modified to increase target site binding specificity, which will decrease off-target editing (A). Tru-sgRNAs are shortened and bind just 17 or 18 nucleotides instead of the typical 20 nucleotide target site. GG-sgRNAs contain two guanines at the 5’ end of the guide, which have been shown to increase binding specificity. Another way to reduce off-target cleavage is with alternative Cas9 protein structures (B). Paired nickases create single strand DNA breaks, thus requiring two different gRNAs to targets close to one another to create a double strand break. Nuclease-dead Cas9 can be fused to the FokI nuclease domain, which requires two nucleases to bind in the correct orientation and distance to dimerize and cleave the DNA.

Figure 13 CRISPR epigenetic regulators. This figure shows different CRISPR-Cas9 methods for activating and repressing genes. Teaching points: The nuclease activity of Cas9 can be abolished by two point mutations (grey ovals). Because Cas9 is easily programmable, researchers use the nuclease dead Cas9 as a shuttle protein to bring different effectors to user-specified sites within the genome. Repression can lead to reduction or loss of protein. Activation can lead to increase in protein expression.

Figure 14 Pooled and Arrayed Screening Strategies. This figure shows the general workflow for a CRISPR-Cas9 knockout screen. Teaching points: CRISPR screens can be performed in pooled or arrayed format. In the pooled format, a pool of gRNAs is delivered via viral transduction to a pool of cells. Then, selective pressure is applied. In a positive selection screen, cells in which gRNAs knockout genes that cause a growth or survival advantage are enriched. In a negative selection screen, cell in which gRNAs knockout genes that cause a growth disadvantage or are lethal will be depleted from the population. Pooled screens are easier to perform, but are more limited in their functional readouts. Arrayed screens allow investigators to screen for more subtle phenotypes that would otherwise be lost in a pooled screen. In an arrayed screen individual gRNAs are applied to individual wells of cells in 96 of 384-well format.

Figure 15 Cell line creation workflow. This figure outlines the steps involved in creating a genetically modified cell line with CRISPR-Cas9. The key points are design and validation of reagents (steps 1-5), delivery of reagents into cells (step 6), and screening and selection of desired modified clone(s) (steps 7-10). The funnel depicts the process from a large population of cells with rare desired modification (green cells) to the identification and expansion of a clonal cell line with the confirmed genome modification.

Figure 16 Animal model creation workflow. This figure outlines the steps involved in creating a genetically modified animal model (in this case, mice) with CRISPR–Cas9. The key points are design and validation of reagents (steps 1-5), delivery of reagents into fertilized oocytes via pronuclear injection (step 6), and screening the resulting pups after birth (steps 9 and 10). Mice that carry the desired modification must be bred to confirm that their offspring also carry the modification. CRISPR–Cas9 technology allows for much more rapid generation of mouse models (1 generation) than previous methods.

Figure 17 Using programmable nucleases for human ex vivo therapy. This figure depicts the steps in human gene editing outside of the patient with programmable nucleases. Cells are removed from the body (A), expanded (B), and treated with gene editing reagents (C) before being reintroduced into the body (D). This reintroduction is called an autologous transplant, and the genetically-modified cells must engraft back into the body in order to have a treatment effect on the patient.

 


Related Articles:

Teaching Material

Contact Editor

Submit a note to the editor about this article by filling in the form below.

* Required Field

How to Cite

Shaina N. Porter, Rachel M. Levine, Shondra M. Pruett‐Miller. A Practical Guide to Genome Editing Using Targeted Nuclease Technologies. Compr Physiol 2019, 9: 665-714. doi: 10.1002/cphy.c180022