Off-target Effects in CRISPR/Cas9-mediated Genome Engineering
2015; Cell Press; Volume: 4; Linguagem: Inglês
10.1038/mtna.2015.37
ISSN2162-2531
AutoresXiaohui Zhang, Louis Y. Tee, Xiaogang Wang, Qunshan Huang, Shihua Yang,
Tópico(s)Innovation and Socioeconomic Development
ResumoCRISPR/Cas9 is a versatile genome-editing technology that is widely used for studying the functionality of genetic elements, creating genetically modified organisms as well as preclinical research of genetic disorders. However, the high frequency of off-target activity (≥50%)—RGEN (RNA-guided endonuclease)-induced mutations at sites other than the intended on-target site—is one major concern, especially for therapeutic and clinical applications. Here, we review the basic mechanisms underlying off-target cutting in the CRISPR/Cas9 system, methods for detecting off-target mutations, and strategies for minimizing off-target cleavage. The improvement off-target specificity in the CRISPR/Cas9 system will provide solid genotype–phenotype correlations, and thus enable faithful interpretation of genome-editing data, which will certainly facilitate the basic and clinical application of this technology. CRISPR/Cas9 is a versatile genome-editing technology that is widely used for studying the functionality of genetic elements, creating genetically modified organisms as well as preclinical research of genetic disorders. However, the high frequency of off-target activity (≥50%)—RGEN (RNA-guided endonuclease)-induced mutations at sites other than the intended on-target site—is one major concern, especially for therapeutic and clinical applications. Here, we review the basic mechanisms underlying off-target cutting in the CRISPR/Cas9 system, methods for detecting off-target mutations, and strategies for minimizing off-target cleavage. The improvement off-target specificity in the CRISPR/Cas9 system will provide solid genotype–phenotype correlations, and thus enable faithful interpretation of genome-editing data, which will certainly facilitate the basic and clinical application of this technology. The CRISPR (clustered, regularly interspaced, short palindromic repeats)/Cas9 system, which is found in diverse bacterial and archaeal species, has been used successfully to edit eukaryotic genomes.1Mali P Yang L Esvelt KM Aach J Guell M DiCarlo JE et al.RNA-guided human genome engineering via Cas9.Science. 2013; 339: 823-826Crossref PubMed Scopus (6231) Google Scholar,2Cong L Ran FA Cox D Lin S Barretto R Habib N et al.Multiplex genome engineering using CRISPR/Cas systems.Science. 2013; 339: 819-823Crossref PubMed Scopus (9635) Google Scholar It now also holds great promise in diverse fields such as animal disease modeling, material science, genetically modified plant technology, biofuel technology, gene therapy, and drug development. In addition, CRISPR/Cas9 technology has substantially accelerated the understanding of functional organization of the genome at the systems level and thus helps to establish solid causal links between genetic variations and biological phenotypes.3Hsu PD Lander ES Zhang F Development and applications of CRISPR-Cas9 for genome engineering.Cell. 2014; 157: 1262-1278Abstract Full Text Full Text PDF PubMed Scopus (3485) Google Scholar,4Doudna JA Charpentier E Genome editing. The new frontier of genome engineering with CRISPR-Cas9.Science. 2014; 346: 1258096Crossref PubMed Scopus (3422) Google Scholar,5Cox DB Platt RJ Zhang F Therapeutic genome editing: prospects and challenges.Nat Med. 2015; 21: 121-131Crossref PubMed Scopus (805) Google Scholar,6Smith C Abalde-Atristain L He C Brodsky BR Braunstein EM Chaudhari P et al.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 (130) Google Scholar However, off-target mutations observed at frequencies greater than the intended mutation, which may cause genomic instability and disrupt the functionality of otherwise normal genes, is still one major concern when applying CRISPR/Cas9 system to biomedical and clinical application.7Mali P Aach J Stranges PB Esvelt KM Moosburner M Kosuri S et al.CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering.Nat Biotechnol. 2013; 31: 833-838Crossref PubMed Scopus (1269) Google Scholar,8Hsu PD Scott DA Weinstein JA Ran FA Konermann S Agarwala V et al.DNA targeting specificity of RNA-guided Cas9 nucleases.Nat Biotechnol. 2013; 31: 827-832Crossref PubMed Scopus (2917) Google Scholar,9Pattanayak V Lin S Guilinger JP Ma E Doudna JA Liu DR High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity.Nat Biotechnol. 2013; 31: 839-843Crossref PubMed Scopus (1037) Google Scholar,10Fu Y Foden JA Khayter C Maeder ML Reyon D Joung JK et al.High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells.Nat Biotechnol. 2013; 31: 822-826Crossref PubMed Scopus (2134) Google Scholar,11Cho SW Kim S Kim Y Kweon J Kim HS Bae S et al.Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases.Genome Res. 2014; 24: 132-141Crossref PubMed Scopus (966) Google Scholar,12Corrigan-Curay J O'Reilly M Kohn DB Cannon PM Bao G Bushman FD et al.Genome editing technologies: defining a path to clinic.Mol Ther. 2015; 23: 796-806Abstract Full Text Full Text PDF PubMed Scopus (82) Google ScholarMechanism of Off-Target Effects In Crispr/Cas9 SystemThe CRISPR/Cas9 system functions as the RNA-based adaptive immune system in bacteria and archaea.13Horvath P Barrangou R CRISPR/Cas, the immune system of bacteria and archaea.Science. 2010; 327: 167-170Crossref PubMed Scopus (1532) Google Scholar The type II CRISPR system, which includes CRISPR-associated nuclease 9 (Cas9), is derived from Streptococcus pyogenes. Native CRISPR system confers resistance to viruses by incorporating short repeats of the viral DNA into the bacterial genome. When a bacterial colony is infected the second time, transcripts of these repeats direct a nuclease to the complementary DNA from the invading virus and thus destroy the viral DNA.14Gasiunas G Barrangou R Horvath P Siksnys V Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria.Proc Natl Acad Sci USA. 2012; 109: E2579-E2586Crossref PubMed Scopus (1581) Google Scholar,15Jinek M Chylinski K Fonfara I Hauer M Doudna JA Charpentier E A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science. 2012; 337: 816-821Crossref PubMed Scopus (8881) Google Scholar To enable its gene-targeting capacity in the eukaryotic cell, the CRISPR/Cas9 system can be reconstituted in mammalian cells using the following three minimal components: Cas9, a specificity-determining CRISPR RNA (crRNA), and an auxiliary trans-activating RNA (tracrRNA).1Mali P Yang L Esvelt KM Aach J Guell M DiCarlo JE et al.RNA-guided human genome engineering via Cas9.Science. 2013; 339: 823-826Crossref PubMed Scopus (6231) Google Scholar,2Cong L Ran FA Cox D Lin S Barretto R Habib N et al.Multiplex genome engineering using CRISPR/Cas systems.Science. 2013; 339: 819-823Crossref PubMed Scopus (9635) Google Scholar,8Hsu PD Scott DA Weinstein JA Ran FA Konermann S Agarwala V et al.DNA targeting specificity of RNA-guided Cas9 nucleases.Nat Biotechnol. 2013; 31: 827-832Crossref PubMed Scopus (2917) Google Scholar The crRNA and tracrRNA duplexes can also be fused to generate a chimeric single-guide RNA (sgRNA). The first ∼20 nucleotides of the sgRNA are complementary to the target DNA sequence, followed by a sequence called the protospacer adjacent motif (PAM), typically NGG.16Mojica FJ Díez-Villaseñor C García-Martínez J Almendros C Short motif sequences determine the targets of the prokaryotic CRISPR defence system.Microbiology. 2009; 155: 733-740Crossref PubMed Scopus (972) Google Scholar,17Sternberg SH Redding S Jinek M Greene EC Doudna JA DNA interrogation by the CRISPR RNA-guided endonuclease Cas9.Nature. 2014; 507: 62-67Crossref PubMed Scopus (1122) Google Scholar,18Anders C Niewoehner O Duerst A Jinek M Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease.Nature. 2014; 513: 569-573Crossref PubMed Scopus (755) Google ScholarAlthough the targeting specificity of Cas9 is believed to be tightly controlled by the 20-nt guide sequence of the sgRNA and the presence of a PAM adjacent to the target sequence in the genome, potential off-target cleavage activity could still occur on DNA sequence with even three to five base pair mismatches in the PAM-distal part of the sgRNA-guiding sequence.2Cong L Ran FA Cox D Lin S Barretto R Habib N et al.Multiplex genome engineering using CRISPR/Cas systems.Science. 2013; 339: 819-823Crossref PubMed Scopus (9635) Google Scholar,7Mali P Aach J Stranges PB Esvelt KM Moosburner M Kosuri S et al.CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering.Nat Biotechnol. 2013; 31: 833-838Crossref PubMed Scopus (1269) Google Scholar,8Hsu PD Scott DA Weinstein JA Ran FA Konermann S Agarwala V et al.DNA targeting specificity of RNA-guided Cas9 nucleases.Nat Biotechnol. 2013; 31: 827-832Crossref PubMed Scopus (2917) Google Scholar,9Pattanayak V Lin S Guilinger JP Ma E Doudna JA Liu DR High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity.Nat Biotechnol. 2013; 31: 839-843Crossref PubMed Scopus (1037) Google Scholar,10Fu Y Foden JA Khayter C Maeder ML Reyon D Joung JK et al.High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells.Nat Biotechnol. 2013; 31: 822-826Crossref PubMed Scopus (2134) Google Scholar Moreover, previous studies have demonstrated that different guide RNA structures can affect the cleavage of on-target and off-target sites.8Hsu PD Scott DA Weinstein JA Ran FA Konermann S Agarwala V et al.DNA targeting specificity of RNA-guided Cas9 nucleases.Nat Biotechnol. 2013; 31: 827-832Crossref PubMed Scopus (2917) Google Scholar Crystal structure studies and single-molecule DNA curtain experiments suggest that while the PAM site is essential for the initiation of Cas9 binding, the seed sequence corresponding to 3′ end of the crRNA complementary recognition sequence, directly adjacent to PAM, is also critical for subsequent Cas9 binding, R-loop formation, and activation of nuclease activities in Cas9.19Nishimasu H Ran FA Hsu PD Konermann S Shehata SI Dohmae N et al.Crystal structure of Cas9 in complex with guide RNA and target DNA.Cell. 2014; 156: 935-949Abstract Full Text Full Text PDF PubMed Scopus (1103) Google Scholar,20Jinek M Jiang F Taylor DW Sternberg SH Kaya E Ma E et al.Structures of Cas9 endonucleases reveal RNA-mediated conformational activation.Science. 2014; 343: 1247997Crossref PubMed Scopus (696) Google ScholarsgRNASgRNA comprises the seed sequence and nonseed sequence (Figure 1). Several initial studies have shown that the 10–12 base pairs adjacent to the PAM (3′ end of the guide RNA), called the "seed sequence", determine Cas9 specificity and is generally more important than the rest of the guide RNA sequences.2Cong L Ran FA Cox D Lin S Barretto R Habib N et al.Multiplex genome engineering using CRISPR/Cas systems.Science. 2013; 339: 819-823Crossref PubMed Scopus (9635) Google Scholar,15Jinek M Chylinski K Fonfara I Hauer M Doudna JA Charpentier E A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science. 2012; 337: 816-821Crossref PubMed Scopus (8881) Google Scholar However, chromatin immunoprecipitation followed by sequencing (ChIP-seq) of DNA bound to catalytically dead Cas9 (dCas9) in murine embryonic stem cells demonstrates that only one to five base pairs of the immunoprecipitated DNA match to the guide region, suggesting that the one to five base pairs of guide region proximal to the PAM mark the true "seed region".21Wu X Scott DA Kriz AJ Chiu AC Hsu PD Dadon DB et al.Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells.Nat Biotechnol. 2014; 32: 670-676Crossref PubMed Scopus (635) Google Scholar These results were also confirmed by Yu Zhang and his colleagues by ChIP-seq analysis of human genome after targeting with CRISPR/Cas922Duan J Lu G Xie Z Lou M Luo J Guo L et al.Genome-wide identification of CRISPR/Cas9 off-targets in human genome.Cell Res. 2014; 24: 1009-1012Crossref PubMed Scopus (103) Google Scholar, and were consistent with the previous observations that base-pairing adjacent to the PAM is critical for DNA targeting.17Sternberg SH Redding S Jinek M Greene EC Doudna JA DNA interrogation by the CRISPR RNA-guided endonuclease Cas9.Nature. 2014; 507: 62-67Crossref PubMed Scopus (1122) Google Scholar,23Jiang W Bikard D Cox D Zhang F Marraffini LA RNA-guided editing of bacterial genomes using CRISPR-Cas systems.Nat Biotechnol. 2013; 31: 233-239Crossref PubMed Scopus (1611) Google Scholar Yet the ChIP-seq assay for detecting off target sites of dCas9 only capture the PAM-proximal binding event but not cleavage event, leading to over-prediction of off-target sites. Pelletier and his colleagues reported that sequences distal to PAM, which possibly triggers a conformational change of Cas9, engage CRISPR/Cas9 DNA target cleavage, and thus using sgRNAs that places equal emphasis on seed sequence and PAM-distal target sequences will lead to lower off-target editing compared to ChIP-seq data only using seed regions.24Cencic R Miura H Malina A Robert F Ethier S Schmeing TM et al.Protospacer adjacent motif (PAM)-distal sequences engage CRISPR Cas9 DNA target cleavage.PLoS One. 2014; 9: e109213Crossref PubMed Scopus (76) Google Scholar The results could be considered to be a supplement to what Sharp and colleagues have reported in their genome-wide binding maps of dCas9 study through ChIP-seq in mouse embryonic stem cells, in which they have revealed well-defined seed region for target binding and very abundant off-target binding sites, however, majority of which do not undergo substantial cleavage by Cas9.21Wu X Scott DA Kriz AJ Chiu AC Hsu PD Dadon DB et al.Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells.Nat Biotechnol. 2014; 32: 670-676Crossref PubMed Scopus (635) Google ScholarThe seed sequence influences the specificity of Cas9-sgRNA binding through multiple potential mechanisms. The sequence of the seed region determines the frequency of a "seed + NGG" in the genome, and controls the effective concentration of the Cas9-sgRNA complex (Cas9 binding or sgRNA abundance and specificity).21Wu X Scott DA Kriz AJ Chiu AC Hsu PD Dadon DB et al.Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells.Nat Biotechnol. 2014; 32: 670-676Crossref PubMed Scopus (635) Google Scholar,25Wang T Wei JJ Sabatini DM Lander ES Genetic screens in human cells using the CRISPR-Cas9 system.Science. 2014; 343: 80-84Crossref PubMed Scopus (1821) Google Scholar Meanwhile, U-rich seeds are likely to result in decreased sgRNA abundance and increased specificity since multiple U's in the sequence can induce termination of sgRNA transcription.21Wu X Scott DA Kriz AJ Chiu AC Hsu PD Dadon DB et al.Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells.Nat Biotechnol. 2014; 32: 670-676Crossref PubMed Scopus (635) Google Scholar,25Wang T Wei JJ Sabatini DM Lander ES Genetic screens in human cells using the CRISPR-Cas9 system.Science. 2014; 343: 80-84Crossref PubMed Scopus (1821) Google Scholar In general, mismatches of the one to five base pairs at the 5′ end of sgRNAs are better tolerated than those at the 3′ end. Single and double mismatches are tolerated to various degrees depending on their position along the guide RNA-DNA interface.10Fu Y Foden JA Khayter C Maeder ML Reyon D Joung JK et al.High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells.Nat Biotechnol. 2013; 31: 822-826Crossref PubMed Scopus (2134) Google Scholar,15Jinek M Chylinski K Fonfara I Hauer M Doudna JA Charpentier E A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science. 2012; 337: 816-821Crossref PubMed Scopus (8881) Google Scholar,23Jiang W Bikard D Cox D Zhang F Marraffini LA RNA-guided editing of bacterial genomes using CRISPR-Cas systems.Nat Biotechnol. 2013; 31: 233-239Crossref PubMed Scopus (1611) Google Scholar It has also been reported that sgRNAs with exceptionally low or high GC content tends to be less active.24Cencic R Miura H Malina A Robert F Ethier S Schmeing TM et al.Protospacer adjacent motif (PAM)-distal sequences engage CRISPR Cas9 DNA target cleavage.PLoS One. 2014; 9: e109213Crossref PubMed Scopus (76) Google Scholar,26Doench JG Hartenian E Graham DB Tothova Z Hegde M Smith I et al.Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation.Nat Biotechnol. 2014; 32: 1262-1267Crossref PubMed Scopus (912) Google Scholar In a study of CRISPR/Cas9-mediated mutagenesis in Drosophila, Ren et al. observed positive correlation between mutagenesis efficiency and GC content of the most proximal region to the PAM sequence of the sgRNAs, based on their evidence that sgRNAs with at least four GCs in the sequence of the six base pairs that are most proximal to the PAM sequence have a heritable mutation (that means mutation generated by effective sgRNA of the CRISPR/Cas9 system from parents is able to pass on to the next generation) rate over 60%, suggesting that effective sgRNAs can be selected according to the GC content of the sequence proximal to PAM.27Ren X Yang Z Xu J Sun J Mao D Hu Y et al.Enhanced specificity and efficiency of the CRISPR/Cas9 system with optimized sgRNA parameters in Drosophila.Cell Rep. 2014; 9: 1151-1162Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar When choosing an appropriate sgRNA sequence, guanine is strongly preferred, and cytosine is strongly unfavorable, as the first base of the seed sequence immediately adjacent to the PAM. Conversely, there is a preference for cytosine, but not guanine, at position 5 that is fifth base proximal to PAM. Adenine is favored in the middle of the sgRNA, and cytosine is disfavored at position 18.21Wu X Scott DA Kriz AJ Chiu AC Hsu PD Dadon DB et al.Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells.Nat Biotechnol. 2014; 32: 670-676Crossref PubMed Scopus (635) Google Scholar,25Wang T Wei JJ Sabatini DM Lander ES Genetic screens in human cells using the CRISPR-Cas9 system.Science. 2014; 343: 80-84Crossref PubMed Scopus (1821) Google Scholar,26Doench JG Hartenian E Graham DB Tothova Z Hegde M Smith I et al.Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation.Nat Biotechnol. 2014; 32: 1262-1267Crossref PubMed Scopus (912) Google Scholar,28Gagnon JA Valen E Thyme SB Huang P Akhmetova L Ahkmetova L et al.Efficient mutagenesis by Cas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs.PLoS One. 2014; 9: e98186Crossref PubMed Scopus (528) Google Scholar (Figure 1) These design principles are probably based upon the theory that guanine-rich sequences can fold into stable noncanonical structures called G-quadruplexes in vivo, which contributes to sgRNA stability.29Moreno-Mateos MA Vejnar CE Beaudoin JD Fernandez JP Mis EK Khokha MK et al.CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo.Nat Methods. 2015; 12: 982-988Crossref PubMed Scopus (621) Google Scholar However, this base preference is largely depending on the target site in many cases,10Fu Y Foden JA Khayter C Maeder ML Reyon D Joung JK et al.High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells.Nat Biotechnol. 2013; 31: 822-826Crossref PubMed Scopus (2134) Google Scholar and the tail sequence of tracrRNA is also very critical for Cas9 activity in vivo.8Hsu PD Scott DA Weinstein JA Ran FA Konermann S Agarwala V et al.DNA targeting specificity of RNA-guided Cas9 nucleases.Nat Biotechnol. 2013; 31: 827-832Crossref PubMed Scopus (2917) Google ScholarPamThe sequence of the PAM also influences the activity of sgRNA (Figure 1). Initial results indicated that NGG (N is A, T, C, or G) is the canonical sequence for the PAM. However, recent studies have suggested that the type II CRISPR system can also use NRG (Figure 1) (where R is G or A) as PAM sequence, albeit with only one-fifth of the binding efficiency compared to NGG. Several studies reported that the NRG sequence is the predominant noncanonical PAM for CRISPR/Cas9-mediated DNA cleavage at the human EMX locus.8Hsu PD Scott DA Weinstein JA Ran FA Konermann S Agarwala V et al.DNA targeting specificity of RNA-guided Cas9 nucleases.Nat Biotechnol. 2013; 31: 827-832Crossref PubMed Scopus (2917) Google Scholar,23Jiang W Bikard D Cox D Zhang F Marraffini LA RNA-guided editing of bacterial genomes using CRISPR-Cas systems.Nat Biotechnol. 2013; 31: 233-239Crossref PubMed Scopus (1611) Google Scholar The binding frequency of each base in the PAM sequence is different. The first nucleotide is the least conserved, with G in nearly 50% of binding sites, while the second position with G in >90% of the binding sites,8Hsu PD Scott DA Weinstein JA Ran FA Konermann S Agarwala V et al.DNA targeting specificity of RNA-guided Cas9 nucleases.Nat Biotechnol. 2013; 31: 827-832Crossref PubMed Scopus (2917) Google Scholar,30Kuscu C Arslan S Singh R Thorpe J Adli M Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease.Nat Biotechnol. 2014; 32: 677-683Crossref PubMed Scopus (538) Google Scholar suggesting that NRG is not the optimal PAM for the designing of CRISPR/Cas9 sequences. Therefore, the exact effect of NRG PAM sequence on DNA cleavage of Cas9 is largely unclear.31Zhang Y Ge X Yang F Zhang L Zheng J Tan X et al.Comparison of non-canonical PAMs for CRISPR/Cas9-mediated DNA cleavage in human cells.Sci Rep. 2014; 4: 5405Crossref PubMed Scopus (148) Google ScholarWhen sgRNAs corresponding to a target DNA site are designed via common CRISPR/Cas9 design tools (Table 1), every sgRNA has its own PAM, typically NGG. However, in practice, alternatively NGG may be not exist corresponding to the effective sgRNA if we want to achieve precise insertion or precise point mutation in the genome, and thus NRG (R=G or A) sequence can be considered as alternative, albeit low cleavage efficiency.31Zhang Y Ge X Yang F Zhang L Zheng J Tan X et al.Comparison of non-canonical PAMs for CRISPR/Cas9-mediated DNA cleavage in human cells.Sci Rep. 2014; 4: 5405Crossref PubMed Scopus (148) Google Scholar In the cases that both NGG and NRG(R=G or A) are not able to provide optimized design of sgRNAs, one may choose the other CRISPR/Cas9 system (Cas9 orthologues, Streptococcus thermophilus Cas9 and Staphylococcusaureus Cas9). When applying these Cas9 orthologues for gene editing, NGA, NAC (Figure 2) can be considered without causing higher off-target effects as compared to wild-type SpCas9.32Kleinstiver BP Prew MS Tsai SQ Topkar VV Nguyen NT Zheng Z et al.Engineered CRISPR-Cas9 nucleases with altered PAM specificities.Nature. 2015; 523: 481-485Crossref PubMed Scopus (1030) Google ScholarTable 1Common CRISPR/Cas9 design toolsNameWebAffiliationCRISPR designhttp://crispr.mit.edu/Massachusetts Institute of TechnologyE-Crispwww.e-crisp.org/E-CRISP/designcrisprDKFZ German Cancer Research CenterCRISPR designhttp://www.broadinstitute.The Broad Institute oftoolorg/mpg/crispr_design/Harvard and MITCas-OFFinderhttp://www.rgenome.netHarvard Medical SchoolCROP-IThttp://www.adlilab.org/CROP-IT/homepage.htmlUniversity of Virginia Open table in a new tab Figure 2Methods for optimal CRISPR/Cas9 system for biomedical and clinical application. (a) Double nicking by RNA-Guided CRISPR Cas9 for enhanced genome-editing specificity. Cas9 protein is shown in orange. Scissors indicate cleavage sites. (b) Fusion of catalytically inactive Cas9 to Fok I nuclease. (c) Delivery of Cas9 protein. cPP, cell-penetrating peptide. The single-letter codes for amino acids are used. C, cysteine; G, glycine; R, arginine; L, leucine. His, Histidine tag; HA, hemagglutinin tag; Mal, maleimide. (d) Alternative PAMs for CIRSPR/Cas9 system. (e) SgRNA design—CRISPR/Cas9 design tools. (f) Method of off-target detection-Degenome-seq. IGV, Integrative Genomics Viewer; WGS, whole-genome sequencing; WT, wild type.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Cas9 Protein and Other FactorsDirect delivery of purified Cas9 protein and sgRNA into cells has been reported to result in reduced off-target effects compared to the delivery of plasmid sequences encoding Cas9 and sgRNA, because Cas9-sgRNA ribonucleoprotein complexes cleave chromosomal DNA almost immediately after delivery and are degraded rapidly in cells.33Kim 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. 2014; 24: 1012-1019Crossref PubMed Scopus (1078) Google Scholar,34Ramakrishna S Kwaku Dad AB Beloor J Gopalappa R Lee SK Kim H Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA.Genome Res. 2014; 24: 1020-1027Crossref PubMed Scopus (443) Google ScholarOff-target effects might be cell-type-specific and highly depending on the integrity of double-stranded breaks (DSBs) repair pathways of particular cell type.22Duan J Lu G Xie Z Lou M Luo J Guo L et al.Genome-wide identification of CRISPR/Cas9 off-targets in human genome.Cell Res. 2014; 24: 1009-1012Crossref PubMed Scopus (103) Google Scholar For example, nuclease off-target effects can occur in transformed human cell lines with dysregulated DSBs repair pathways, while whole-genome sequencing of healthy human pluripotent stem cell clones with relatively intact DSBs repairing capability have revealed very few off-target mutations attributable to the nucleases.35Smith C Gore A Yan W Abalde-Atristain L Li Z He C et al.Whole-genome sequencing analysis reveals high specificity of CRISPR/Cas9 and TALEN-based genome editing in human iPSCs.Cell Stem Cell. 2014; 15: 12-13Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar,36Veres A Gosis BS Ding Q Collins R Ragavendran A Brand H et al.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. 2014; 15: 27-30Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar Furthermore, it has been reported that methylation of DNA at CpG sites may impede the binding efficiency of Cas9 in cells, and small molecules that enhance CRISPR genome editing by promoting precise genome editing via homology-directed repair (e.g., L755507, a 73-adrenergic receptor agonist, and Brefeldin A, an inhibitor of intracellular protein transport from the ER to the Golgi apparatus) or sequence-specific gene knockout via nonhomologous end-joining (NHEJ) (e.g., azidothymidineorTrifluridine) in pluripotent stem cells are being studied.21Wu X Scott DA Kriz AJ Chiu AC Hsu PD Dadon DB et al.Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells.Nat Biotechnol. 2014; 32: 670-676Crossref PubMed Scopus (635) Google Scholar,22Duan J Lu G Xie Z Lou M Luo J Guo L et al.Genome-wide identification of CRISPR/Cas9 off-targets in human genome.Cell Res. 2014; 24: 1009-1012Crossref PubMed Scopus (103) Google Scholar,30Kuscu C Arslan S Singh R Thorpe J Adli M Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease.Nat Biotechnol. 2014; 32: 677-683Crossref PubMed Scopus (538) Google Scholar,37Yu C Liu Y Ma T Liu K Xu S Zhang Y et al.Small molecules enhance CRISPR genome editing in pluripotent stem cells.Cell Stem Cell. 2015; 16: 142-147Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar Moreover, toward the goal of achieving more efficient genome editing with CRISPR/Cas9, more explorative studies by employing epigenetic, DSBs repairing pathways modulators, such as small molecules or RNAi strategies that can further enhance or inhibit specific genome-editing pathways of Cas9 via homology-directed repair or NHEJ, need to be more extensively examined.In summary, the seed sequence and the PAM, which are indispensable components of CRISPR/Cas9, need to be carefully designed. Moreover, the application of purified Cas9 protein as well as modulatory small molecules of DSBs pathways, which influence both on-target efficiency and off-target specificity, should be considered in individual application.Methods of Off-Target DetectionDetecting off-target sites in a highly sensitive and comprehensive manner remains a key challenge in the field of gene editing.38Gabriel R von Kalle C Schmidt M Mapping the precision of genome editing.Nat Biotechnol. 2015; 33: 150-152Crossref PubMed Scopus (30) Google Scholar The T7 endonuclease I assay was initially used to detect off-target mutations, but this assay suffers poor sensitivity (it cannot detect off-target mutations that occur at frequencies <1%), and it is neither practical nor cost-effective for large-scale screening.39Cho SW Kim S Kim JM Kim JS Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease.Nat Biotechnol. 2013; 31: 230-232Crossref PubMed Scopus (1343) Google Scholar,40Kim HJ Lee HJ Kim H Cho SW Kim JS Targeted genome editing in human cells with zinc finger nucleases constructed via modular assembly.Genome Res. 2009; 19: 1279-1288Crossref PubMed Scopus (330) Google Scholar Various advanced methods for off-targeting detecting including deep sequencing (measure off-target mutations at frequencies ranging from 0.01 to 0.1%11Cho SW Kim S Kim Y Kweon J Kim HS Bae S et al.Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases.Genome Res. 2014; 24: 132-141Crossref PubMed Scopus (966) Google Scholar), web-based predict
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