Single-Base Resolution: Increasing the Specificity of the CRISPR-Cas System in Gene Editing
Authors: Roy Rabinowitz, Daniel Offen
Journal: Molecular Therapy (2020)

The CRISPR-Cas system holds great promise in the treatment of diseases caused by genetic variations. The Cas protein, an RNA-guided programmable nuclease, generates a double-strand break at precise genomic loci. However, the use of the clustered regularly interspersed short palindromic repeats (CRISPR)-Cas system to distinguish between single-nucleotide variations is challenging. The promiscuity of the guide RNA (gRNA) and its mismatch tolerance make allele-specific targeting an elusive goal. This review presents a meta-analysis of previous studies reporting position-dependent mismatch tolerance within the gRNA. We also examine the conservativity of the seed sequence, a region within the gRNA with stringent sequence dependency, and propose the existence of a subregion within the seed sequence with a higher degree of specificity. In addition, we summarize the reports on high-fidelity Cas nucleases with improved specificity and compare the standard gRNA design methodology to the single-nucleotide polymorphism (SNP)-derived protospacer adjacent motif (PAM) approach, an alternative method for allele-specific targeting. The combination of the two methods may be advantageous in designing CRISPR-based therapeutics and diagnostics for heterozygous patients. The CRISPR-Cas system holds great promise in the treatment of diseases caused by genetic variations. The Cas protein, an RNA-guided programmable nuclease, generates a double-strand break at precise genomic loci. However, the use of the clustered regularly interspersed short palindromic repeats (CRISPR)-Cas system to distinguish between single-nucleotide variations is challenging. The promiscuity of the guide RNA (gRNA) and its mismatch tolerance make allele-specific targeting an elusive goal. This review presents a meta-analysis of previous studies reporting position-dependent mismatch tolerance within the gRNA. We also examine the conservativity of the seed sequence, a region within the gRNA with stringent sequence dependency, and propose the existence of a subregion within the seed sequence with a higher degree of specificity. In addition, we summarize the reports on high-fidelity Cas nucleases with improved specificity and compare the standard gRNA design methodology to the single-nucleotide polymorphism (SNP)-derived protospacer adjacent motif (PAM) approach, an alternative method for allele-specific targeting. The combination of the two methods may be advantageous in designing CRISPR-based therapeutics and diagnostics for heterozygous patients. The clustered regularly interspaced short palindromic repeats (CRISPR) system is a microbial adaptive immune system repurposed as a powerful genome-editing tool. A variety of bacteria and archaea contains an endogenous RNA-based adaptive immune system that can degrade the nucleic acids of invading phages and plasmids. These systems consist of CRISPR genes that produce RNA components and CRISPR-associated (Cas) genes that encode protein components. The CRISPR RNAs (crRNAs) contain short stretches of homology to specific viruses and plasmids (spacers) and act as guides to direct Cas nucleases to degrade the complementary nucleic acids of the relevant pathogen.1Jinek M. Chylinski K. Fonfara I. Hauer M. Doudna J.A. Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science. 2012; 337: 816-821Crossref PubMed Scopus (7756) Google Scholar The protospacer adjacent motif (PAM) is a short sequence, adjacent to the crRNA-targeted sequence, on the target DNA, and has an essential role in DNA binding and cleavage.2Cong L. Ran F.A. Cox D. Lin S. Barretto R. Habib N. Hsu P.D. Wu X. Jiang W. Marraffini L.A. Zhang F. Multiplex genome engineering using CRISPR/Cas systems.Science. 2013; 339: 819-823Crossref PubMed Scopus (8755) Google Scholar The RNA-guided Cas9 protein has two properties that distinguish it as a promising genome-editing tool, namely, its ability to specifically bind unique double-stranded DNA sequences, as directed by the guide RNA (gRNA), and the capacity to then make a DNA double-strand break (DSB). Once the DSB has occurred, cellular repair mechanisms are recruited to restore DNA integrity. Non-homologous end joining (NHEJ) is a predominant and generally accurate error-free cell DNA-repair pathway that permits a low rate of errors in order to balance genetic stability and genetic diversity.3Bétermier M. Bertrand P. Lopez B.S. Is non-homologous end-joining really an inherently error-prone process?.PLoS Genet. 2014; 10: e1004086Crossref PubMed Scopus (230) Google Scholar As long as the NHEJ results in precise restoration of the DSB, Cas9 is able to re-cleave the repair, since its gRNA matches the target DNA in the presence of an intact PAM. However, once the NHEJ machinery stumbles, and an indel (insertion/deletion) is installed in place of the original sequence, the Cas9 target sequence is disrupted, and the newly formed indel remains embedded within the genome. As a result, in terms of genome engineering, NHEJ is considered an error-prone indel-forming pathway. Researchers exploit the error-prone property of NHEJ to randomly install indels, which in most cases, give rise to a frameshift mutation that leads to knockout of the target gene.4Wu Y. Liang D. Wang Y. Bai M. Tang W. Bao S. Yan Z. Li D. Li J. Correction of a genetic disease in mouse via use of CRISPR-Cas9.Cell Stem Cell. 2013; 13: 659-662Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar, 5Wang H. Yang H. Shivalila C.S. Dawlaty M.M. Cheng A.W. Zhang F. Jaenisch R. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering.Cell. 2013; 153: 910-918Abstract Full Text Full Text PDF PubMed Scopus (2374) Google Scholar, 6Shalem O. Sanjana N.E. Hartenian E. Shi X. Scott D.A. Mikkelson T. Heckl D. Ebert B.L. Root D.E. Doench J.G. Zhang F. Genome-scale CRISPR-Cas9 knockout screening in human cells.Science. 2014; 343: 84-87Crossref PubMed Scopus (2691) Google Scholar Homology-directed repair (HDR) is a less-frequently used cellular repair pathway, by which the cell employs a DNA template and homologous recombination machinery to repair the damaged DNA. Among the repertoire of DNA DSB repair pathways, HDR is considered the only one that can be relied upon to provide a precise outcome. However, the low frequency of HDR compared to NHEJ poses a challenge for gene editing.7Shrivastav M. De Haro L.P. Nickoloff J.A. Regulation of DNA double-strand break repair pathway choice.Cell Res. 2008; 18: 134-147Crossref PubMed Scopus (906) Google Scholar, 8Ran F.A. Hsu P.D. Wright J. Agarwala V. Scott D.A. Zhang F. Genome engineering using the CRISPR-Cas9 system.Nat. Protoc. 2013; 8: 2281-2308Crossref PubMed Scopus (5410) Google Scholar, 9Hou Z. Zhang Y. Propson N.E. Howden S.E. Chu L.-F. Sontheimer E.J. Thomson J.A. Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis.Proc. Natl. Acad. Sci. USA. 2013; 110: 15644-15649Crossref PubMed Scopus (437) Google Scholar Single-nucleotide polymorphisms (SNPs) account for 58% of the disease-associated genetic variations in humans.10Rees H.A. Liu D.R. Base editing: precision chemistry on the genome and transcriptome of living cells.Nat. Rev. Genet. 2018; 19: 770-788Crossref PubMed Scopus (504) Google Scholar Gene therapies designed to correct any consequent pathology require accurate allele-specific targeting in order to maintain an intact copy of the wild-type (WT) or a well-functioning allele. Such precise gene editing can be used to correct mutations or introduce protective mutations and to study point mutation-associated conditions. However, targeting the pathogenic allele while preserving the WT allele is challenging due to specificity properties of the Cas9:gRNA complex. This review describes the specificity of the CRISPR system and the efforts that have been made to develop highly specific Cas variants and allele-specific targeting in order to overcome the difficulties. The term specificity is used herein to describe two issues: targeting the desired allele and reducing off-target activity. Both derive from a common source, namely, the resolution in which Cas enzymes distinguish similar sequences. In principle, single base substitutions should be simpler for gene editing than other genetic variations, such as copy number loss or gain, or large deletions or insertions. However, small differences, such as those represented by SNPs, may pose a challenge to allele-specific targeting due to the tolerance of the CRISPR-Cas system to some degree of mismatch between the gRNA and the target DNA. In order to better understand the specificity characteristics of the Cas9 enzyme from Streptococcus pyogenes (SpCas9), we review the fundamental studies in the field. Jinek et al.1Jinek M. Chylinski K. Fonfara I. Hauer M. Doudna J.A. Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science. 2012; 337: 816-821Crossref PubMed Scopus (7756) Google Scholar examined the effect of gRNA:DNA mismatches and reported that the six positions in the 5′ terminal of the gRNA could tolerate mismatches. Thus, up to six mismatches in the PAM-distal region did not disrupt DNA cleavage. In contrast, single mismatches in the PAM-proximal region did abolish cleavage.1Jinek M. Chylinski K. Fonfara I. Hauer M. Doudna J.A. Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science. 2012; 337: 816-821Crossref PubMed Scopus (7756) Google Scholar While investigating the gRNA:DNA tolerance to mismatches, Cong et al.2Cong L. Ran F.A. Cox D. Lin S. Barretto R. Habib N. Hsu P.D. Wu X. Jiang W. Marraffini L.A. Zhang F. Multiplex genome engineering using CRISPR/Cas systems.Science. 2013; 339: 819-823Crossref PubMed Scopus (8755) Google Scholar found that a single mismatch within the 11 positions upstream of the PAM (PAM-proximal region) completely disrupted DNA cleavage and described the nine PAM-distal bases as tolerant positions. Later, Hsu et al.11Hsu P.D. Scott D.A. Weinstein J.A. Ran F.A. Konermann S. Agarwala V. Li Y. Fine E.J. Wu X. Shalem O. et al.DNA targeting specificity of RNA-guided Cas9 nucleases.Nat. Biotechnol. 2013; 31: 827-832Crossref PubMed Scopus (2676) Google Scholar described the specificity and tolerance of mismatches within the gRNA and reported varying degrees of specificity within the 20 bases that constitute the spacer sequence. Consistent with previous studies, they reported that mismatches within the PAM-distal region are more tolerated and defined a range of 8 to 14 bases in the PAM-proximal region as the seed sequence that provides specificity. Hence, mismatches within the seed sequence lead to poor performance by Cas9.11Hsu P.D. Scott D.A. Weinstein J.A. Ran F.A. Konermann S. Agarwala V. Li Y. Fine E.J. Wu X. Shalem O. et al.DNA targeting specificity of RNA-guided Cas9 nucleases.Nat. Biotechnol. 2013; 31: 827-832Crossref PubMed Scopus (2676) Google Scholar These results were supported by Doench et al.,12Doench J.G. Fusi N. Sullender M. Hegde M. Vaimberg E.W. Donovan K.F. Smith I. Tothova Z. Wilen C. Orchard R. et al.Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9.Nat. Biotechnol. 2016; 34: 184-191Crossref PubMed Scopus (1397) Google Scholar who performed a similar mismatch analysis assay to determine the effects of the mismatch position and type of mismatched bases. Data from both studies revealed that a rG:dT mismatch (where T appears in the target DNA with G instead of A in the complementary position in the gRNA) is the most tolerated mismatch in the 20-nucleotide (nt) spacer.12Doench J.G. Fusi N. Sullender M. Hegde M. Vaimberg E.W. Donovan K.F. Smith I. Tothova Z. Wilen C. Orchard R. et al.Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9.Nat. Biotechnol. 2016; 34: 184-191Crossref PubMed Scopus (1397) Google Scholar In addition, both studies support the seed-sequence consensus, as they observed a trend to increased sensitivity to mismatches across the PAM region. A comparison of Cas9 activity on very similar off-target sequences, differing by a single nucleotide, was carried out by Cho et al.13Cho S.W. Kim S. Kim Y. Kweon J. Kim H.S. Bae S. Kim J.-S. Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases.Genome Res. 2014; 24: 132-141Crossref PubMed Scopus (896) Google Scholar In contrast to the results of previous studies, they concluded that the enzyme could not discriminate between single-base mismatches in either the seed sequence or the non-seed sequence. Whereas a mismatch in the second position nearly abolished Cas activity (1.6% indels), the ninth position exhibited a higher tolerance with nearly the same editing rate as the perfect-match target DNA.13Cho S.W. Kim S. Kim Y. Kweon J. Kim H.S. Bae S. Kim J.-S. Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases.Genome Res. 2014; 24: 132-141Crossref PubMed Scopus (896) Google Scholar Smith et al.14Smith C. Abalde-Atristain L. He C. Brodsky B.R. Braunstein E.M. Chaudhari P. Jang Y.-Y. Cheng L. Ye Z. Efficient and allele-specific genome editing of disease loci in human iPSCs.Mol. Ther. 2015; 23: 570-577Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar demonstrated allele-specific gene editing in heterozygous human induced pluripotent stem cells (iPSCs) carrying mutations in either the JAK2 or SERPINA1 genes. The disease-associated SNP was introduced at the 5th position of the spacer and yielded allele-specific editing in most cases. Some gRNAs also achieved significant DNA cleavage in the WT allele.14Smith C. Abalde-Atristain L. He C. Brodsky B.R. Braunstein E.M. Chaudhari P. Jang Y.-Y. Cheng L. Ye Z. Efficient and allele-specific genome editing of disease loci in human iPSCs.Mol. Ther. 2015; 23: 570-577Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar A GFP disruption assay was used by Fu et al.15Fu Y. Foden J.A. Khayter C. Maeder M.L. Reyon D. Joung J.K. Sander J.D. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells.Nat. Biotechnol. 2013; 31: 822-826Crossref PubMed Scopus (1972) Google Scholar and demonstrated inconsistent Cas9 activity at three different sites of the EGFP gene. Multiple gRNAs for each site were designed, each with a mismatch in a different position, in order to prepare a specificity profile for SpCas9. The results at one site were consistent with the seed-sequence consensus, since the 11 PAM-proximal positions were sensitive to mismatches, although low levels of Cas9 activity were still detected. However, at the other two sites, Cas9 activity was preserved after mismatches in most of the gRNA positions. Interestingly, the mismatch-sensitive positions were different for each site.15Fu Y. Foden J.A. Khayter C. Maeder M.L. Reyon D. Joung J.K. Sander J.D. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells.Nat. Biotechnol. 2013; 31: 822-826Crossref PubMed Scopus (1972) Google Scholar This genomic context-dependent mismatch sensitivity was also observed by Capon et al.,16Capon S.J. Baillie G.J. Bower N.I. da Silva J.A. Paterson S. Hogan B.M. Simons C. Smith K.A. Utilising polymorphisms to achieve allele-specific genome editing in zebrafish.Biol. Open. 2017; 6: 125-131Crossref PubMed Scopus (11) Google Scholar who examined the allele-specificity property of SpCas9 in zebrafish. Two genomically distinct strains, qWIK and AB, were crossed to form a zebrafish with heterozygous polymorphisms. Nineteen gRNAs were then designed to specifically target SNPs originating from the qWIK strain. SNPs were selected to test 13 positions of the gRNA, and all 19 gRNAs were designed to target the qWIK alleles. The results revealed that 14 of the gRNAs displayed a general preference for DNA cleavage when there was a perfect complementarity between the gRNA and target DNA. Out of the five others, four gRNAs evinced no allelic preference, and one actually displayed a preference in favor of the AB allele. Among the 14 allele-specific gRNAs, most generated indels at low frequency in the AB alleles. Interestingly, out of six positions (positions 1, 6, 10, 13, 15, and 18), which were independently tested by two different SNPs, three (positions 1, 6, and 10) yielded inconsistent results, indicating the presence of sequence-dependent sensitivity.16Capon S.J. Baillie G.J. Bower N.I. da Silva J.A. Paterson S. Hogan B.M. Simons C. Smith K.A. Utilising polymorphisms to achieve allele-specific genome editing in zebrafish.Biol. Open. 2017; 6: 125-131Crossref PubMed Scopus (11) Google Scholar Anderson et al.17Anderson E.M. Haupt A. Schiel J.A. Chou E. Machado H.B. Strezoska Ž. Lenger S. McClelland S. Birmingham A. Vermeulen A. Smith A.v. Systematic analysis of CRISPR-Cas9 mismatch tolerance reveals low levels of off-target activity.J. Biotechnol. 2015; 211: 56-65Crossref PubMed Scopus (80) Google Scholar performed a reporter assay to test gRNA tolerance for single mismatches at a given position. They reported consistent results, suggesting a seed sequence in positions 1–10, as well as a less-restricted region in positions 11–20, where single mismatches had a minimal influence on Cas9 activity. The reporter assay was performed using two crRNA sequences, which each displayed a varying degree of mismatch tolerance. Whereas mismatches in the seed sequence generally disrupted Cas9 activity, non-seed-sequence positions were highly tolerant of mismatches without a consistent position pattern between the two gRNAs.17Anderson E.M. Haupt A. Schiel J.A. Chou E. Machado H.B. Strezoska Ž. Lenger S. McClelland S. Birmingham A. Vermeulen A. Smith A.v. Systematic analysis of CRISPR-Cas9 mismatch tolerance reveals low levels of off-target activity.J. Biotechnol. 2015; 211: 56-65Crossref PubMed Scopus (80) Google Scholar In order to achieve a comprehensive picture of the specificity at each position of the gRNA, we conducted a meta-analysis of datasets from six studies that examined the specificity profile of SpCas9.11Hsu P.D. Scott D.A. Weinstein J.A. Ran F.A. Konermann S. Agarwala V. Li Y. Fine E.J. Wu X. Shalem O. et al.DNA targeting specificity of RNA-guided Cas9 nucleases.Nat. Biotechnol. 2013; 31: 827-832Crossref PubMed Scopus (2676) Google Scholar, 12Doench J.G. Fusi N. Sullender M. Hegde M. Vaimberg E.W. Donovan K.F. Smith I. Tothova Z. Wilen C. Orchard R. et al.Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9.Nat. Biotechnol. 2016; 34: 184-191Crossref PubMed Scopus (1397) Google Scholar, 13Cho S.W. Kim S. Kim Y. Kweon J. Kim H.S. Bae S. Kim J.-S. Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases.Genome Res. 2014; 24: 132-141Crossref PubMed Scopus (896) Google Scholar,15Fu Y. Foden J.A. Khayter C. Maeder M.L. Reyon D. Joung J.K. Sander J.D. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells.Nat. Biotechnol. 2013; 31: 822-826Crossref PubMed Scopus (1972) Google Scholar, 16Capon S.J. Baillie G.J. Bower N.I. da Silva J.A. Paterson S. Hogan B.M. Simons C. Smith K.A. Utilising polymorphisms to achieve allele-specific genome editing in zebrafish.Biol. Open. 2017; 6: 125-131Crossref PubMed Scopus (11) Google Scholar, 17Anderson E.M. Haupt A. Schiel J.A. Chou E. Machado H.B. Strezoska Ž. Lenger S. McClelland S. Birmingham A. Vermeulen A. Smith A.v. Systematic analysis of CRISPR-Cas9 mismatch tolerance reveals low levels of off-target activity.J. Biotechnol. 2015; 211: 56-65Crossref PubMed Scopus (80) Google Scholar The specificity in the described data was measured as cleavage efficiency of a mismatched gRNA compared to a perfect-match gRNA. Thus, low values indicate high specificity and vice versa. As mentioned above, specificity profiling was conducted by more studies other than the six depicted herein. The primary data of those studies, which were not included in the meta-analysis, were not available. Importantly, although data collection and sample size differed, when the reported results in each study were normalized to the appropriate baseline, consistent trends were observed despite the differences in methodologies and experimental models. High-throughput analysis-derived data are more accurate and impactful than analyses of one or two gRNAs per position. Nonetheless, small sample-size studies have other advantages, such as reporting the specificity in different models and genomic contexts. The seed sequence defined as the ten PAM proximal positions was clearly distinguishable from the ten PAM distal positions. Notably, we could also identify a subregion of the seed sequence from positions 2–8, which had a greater sensitivity to mismatches than the other seed-sequence positions (1, 9, and 10). Indeed, after normalizing and dividing the activity levels of Cas9 per position into quartiles, the two lower quartiles (Q1 and Q2) that represent the highest specificity were found to include the ten positions of the seed sequence, whereas the upper quartiles (Q3 and Q4) comprised the ten PAM-distal positions (Figure 1A). The aforementioned studies provide a better understanding of the seed sequence and the nature of its specificity and mismatch sensitivity. The varying results reported in each study (Figure 1B) emphasize the importance of using a standardized method to measure gRNA specificity and Cas9 activity. The discrepancies between the studies may be attributed, at least partially, to the diverse methods used to measure Cas9 activity (Table 1). In this context, different methods of detecting indels have been shown to produce varying results.18Vouillot L. Thélie A. Pollet N. Comparison of T7E1 and surveyor mismatch cleavage assays to detect mutations triggered by engineered nucleases.G3 (Bethesda). 2015; 5: 407-415Crossref PubMed Scopus (194) Google Scholar, 19Cradick T.J. Fine E.J. Antico C.J. Bao G. CRISPR/Cas9 systems targeting β-globin and CCR5 genes have substantial off-target activity.Nucleic Acids Res. 2013; 41: 9584-9592Crossref PubMed Scopus (430) Google Scholar, 20Zischewski J. Fischer R. Bortesi L. Detection of on-target and off-target mutations generated by CRISPR/Cas9 and other sequence-specific nucleases.Biotechnol. Adv. 2017; 35: 95-104Crossref PubMed Scopus (153) Google Scholar The single guide RNA (sgRNA) design strategy of Cho et al.13Cho S.W. Kim S. Kim Y. Kweon J. Kim H.S. Bae S. Kim J.-S. Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases.Genome Res. 2014; 24: 132-141Crossref PubMed Scopus (896) Google Scholar and Capon et al.16Capon S.J. Baillie G.J. Bower N.I. da Silva J.A. Paterson S. Hogan B.M. Simons C. Smith K.A. Utilising polymorphisms to achieve allele-specific genome editing in zebrafish.Biol. Open. 2017; 6: 125-131Crossref PubMed Scopus (11) Google Scholar provides a higher level of confidence regarding the activity of a certain gRNA. Their use of the same gRNA for two similar sequences, where one was a perfect match, and the other differed by a single nucleotide, allowed them to isolate the effect of the gRNA variable. The lower activity on the mismatched sequence was shown to be due to the mismatch itself and not to the ability of a particular gRNA to induce DNA cleavage. However, the small sample size in both studies may account for the different trends observed for some positions. Most studies target a specific DNA region and design multiple gRNAs, each with a mismatch relative to the target sequence in a different position in order to map the full range of mismatch tolerance positions. However, positive controls are not available for all of the tested gRNAs. Moreover, the genomic context may contribute to the disparate results of the different gRNAs checked. The finding of inconsistent results at different target sites has repeated itself in several studies and in between studies. Taken together, these data suggest a position and sequence-dependent mismatch tolerance that is in apparent conflict with the seed-sequence specificity consensus. Another high-throughput analysis study describes the specificity profile of SpCas9 compared to two synthetic variants, xCas9 and SpCas9-NG, with altered PAM requirement. Due to the lack of primary numeric data, this study was not included in the meta-analysis. However, they report results consistent with the seed-sequence consensus and provide further evidence of a highly specific subregion within the seed sequence.21Kim H.K. Lee S. Kim Y. Park J. Min S. Choi J.W. Huang T.P. Yoon S. Liu D.R. Kim H.H. High-throughput analysis of the activities of xCas9, SpCas9-NG and SpCas9 at matched and mismatched target sequences in human cells.Nat. Biomed. Eng. 2020; 4: 111-124Crossref PubMed Scopus (38) Google ScholarTable 1A Summary Table of the Compared StudiesAuthorData Collection MethodSample Size (gRNAs per Position)Experimental ModelCommentsHsu et al.11Hsu P.D. Scott D.A. Weinstein J.A. Ran F.A. Konermann S. Agarwala V. Li Y. Fine E.J. Wu X. Shalem O. et al.DNA targeting specificity of RNA-guided Cas9 nucleases.Nat. Biotechnol. 2013; 31: 827-832Crossref PubMed Scopus (2676) Google Scholardeep sequencing3 per locus. 4 loci within the EMX1 gene were targeted. A profile of all 12 possible substitutions per position was generated.HEK293FT cells−Doench et al.12Doench J.G. Fusi N. Sullender M. Hegde M. Vaimberg E.W. Donovan K.F. Smith I. Tothova Z. Wilen C. Orchard R. et al.Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9.Nat. Biotechnol. 2016; 34: 184-191Crossref PubMed Scopus (1397) Google Scholarflow cytometry3 per locus. Multiple loci within the CD33 gene were targeted. A profile of all 12 possible substitutions per position was generated.MOLM13 cellssgRNAs were designed to target CD33. Cas activity was measured as CD33 knockout percentage by flow cytometry.Cho et al.13Cho S.W. Kim S. Kim Y. Kweon J. Kim H.S. Bae S. Kim J.-S. Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases.Genome Res. 2014; 24: 132-141Crossref PubMed Scopus (896) Google Scholardeep sequencing1K562 cellsoff-target analysis by comparing two similar genomic lociFu et al.15Fu Y. Foden J.A. Khayter C. Maeder M.L. Reyon D. Joung J.K. Sander J.D. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells.Nat. Biotechnol. 2013; 31: 822-826Crossref PubMed Scopus (1972) Google ScholarGFP disruption assay3U2OS.EGFP cellsthree sgRNAs targeting different sites within the EGFP geneCapon et al.16Capon S.J. Baillie G.J. Bower N.I. da Silva J.A. Paterson S. Hogan B.M. Simons C. Smith K.A. Utilising polymorphisms to achieve allele-specific genome editing in zebrafish.Biol. Open. 2017; 6: 125-131Crossref PubMed Scopus (11) Google Scholardeep sequencing1 or 2zebrafish embryos derived from a cross between the qWIK and AB strainsThe two strains have certain SNPs in different genes. The sgRNAs were designed to target only the qWIK SNPs.Anderson et al.17Anderson E.M. Haupt A. Schiel J.A. Chou E. Machado H.B. Strezoska Ž. Lenger S. McClelland S. Birmingham A. Vermeulen A. Smith A.v. Systematic analysis of CRISPR-Cas9 mismatch tolerance reveals low levels of off-target activity.J. Biotechnol. 2015; 211: 56-65Crossref PubMed Scopus (80) Google ScholarUbi-GFP U2OS-Cas9 EGFP fluorescence assay2Ubi-GFP U2OS-Cas9 cells stably express EGFP fused to a non-cleavable ubiquitin moiety. The EGFP is being degraded constitutively through the proteosome pathway.Two gRNAs were designed to target components of the proteosome (either VCP or PSMD7). Disruption of the proteosome complex leads to accumulation of EGFP in the cells. For each target, 20 gRNAs carrying single mismatches were designed and compared to a perfect match gRNA. Open table in a new tab SpCas9 is the most studied and best characterized Cas ortholog with extensive information available about its specificity profile, as well as on-target efficiency and off-target activity. However, other natural Cas enzymes, such as Staphylococcus aureus Cas9 (SaCas9) and Cas12a enzymes (e.g., Acidaminococcus sp. [AsCas12a] and Lachnospiraceae bacterium [LbCas12a]), previously known as Cpf1, have unique properties that may make them preferable to SpCas9 in certain circumstances. SaCas9, as well as Staphylococcus auricularis (SauriCas9)22Hu Z. Wang S. Zhang C. Gao N. Li M. Wang D. Wang D. Liu D. Liu H. Ong S.G. et al.A compact Cas9 ortholog from Staphylococcus Auricularis (SauriCas9) expands the DNA targeting scope.PLoS Biol. 2020; 18: e3000686Crossref PubMed Scopus (1) Google Scholar and the viral CasΦ23Pausch P. Al-Shayeb B. Bisom-Rapp E. Tsuchida C.A. Li Z. Cress B.F. Knott G.J. Jacobsen S.E. Banfield J.F. Doudna J.A. CRISPR-CasΦ from huge phages is a hypercompact genome editor.Science. 2020; 369: 333-337Crossref PubMed Scopus (108) Google Scholar are more compact and therefore fit in adeno-associated virus (AAV) vectors, making them potential candidates for in vivo delivery. In the case of SaCas9, the PAM sequence also differs from the G-rich PAM of SpCas9 (NGG), as it recognizes NNGRRT for a PAM,24Ran F.A. Cong L. Yan W.X. Scott D.A. Gootenberg J.S. Kriz A.J. Zetsche B. Shalem O. Wu X. Makarova K.S. et al.In vivo genome editing using Staphylococcus aureus Cas9.Nature. 2015; 520: 186-191Crossref PubMed Scopus (1502) Google Scholar Tycko et al.25Tycko J. Barrera L.A. Huston N.C. Friedland A.E. Wu X. Gootenberg J.S. Abudayyeh O.O. Myer V.E. Wilson C.J. Hsu P.D. Pairwise library screen systematically interrogates Staphylococcus aureus Cas9 specificity in human cells.Nat. Commun. 2018; 9: 2962Crossref PubMed Scopus (13) Google Scholar performed a series of specificity profiling experiments