Profiling single-guide RNA specificity reveals a mismatch sensitive core sequence
Targeting specificity is an essential issue in the development of CRISPR-Cas technology. Using a luciferase activation assay, off-target cleavage activity of sgRNA was systematically investigated on single nucleotide-mismatched targets. In addition to confirming that PAM-proximal mismatches are less...
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| Veröffentlicht in: | Scientific reports 2017-01, Vol.7 (1), p.40638-40638, Article 40638 |
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| creator | Zheng, Ting Hou, Yingzi Zhang, Pingjing Zhang, Zhenxi Xu, Ying Zhang, Letian Niu, Leilei Yang, Yi Liang, Da Yi, Fan Peng, Wei Feng, Wenjian Yang, Ying Chen, Jianxin Zhu, York Yuanyuan Zhang, Li-He Du, Quan |
| description | Targeting specificity is an essential issue in the development of CRISPR-Cas technology. Using a luciferase activation assay, off-target cleavage activity of sgRNA was systematically investigated on single nucleotide-mismatched targets. In addition to confirming that PAM-proximal mismatches are less tolerated than PAM-distal mismatches, our study further identified a “core” sequence that is highly sensitive to target-mismatch. This sequence is of 4-nucleotide long, located at +4 to +7 position upstream of PAM, and positioned in a steric restriction region when assembled into Cas9 endonuclease. Our study also found that, single or multiple target mismatches at this region abolished off-target cleavage mediated by active sgRNAs, thus proposing a principle for gene-specific sgRNA design. Characterization of a mismatch sensitive “core” sequence not only enhances our understanding of how this elegant system functions, but also facilitates our efforts to improve targeting specificity of a sgRNA. |
| doi_str_mv | 10.1038/srep40638 |
| format | Article |
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Using a luciferase activation assay, off-target cleavage activity of sgRNA was systematically investigated on single nucleotide-mismatched targets. In addition to confirming that PAM-proximal mismatches are less tolerated than PAM-distal mismatches, our study further identified a “core” sequence that is highly sensitive to target-mismatch. This sequence is of 4-nucleotide long, located at +4 to +7 position upstream of PAM, and positioned in a steric restriction region when assembled into Cas9 endonuclease. Our study also found that, single or multiple target mismatches at this region abolished off-target cleavage mediated by active sgRNAs, thus proposing a principle for gene-specific sgRNA design. Characterization of a mismatch sensitive “core” sequence not only enhances our understanding of how this elegant system functions, but also facilitates our efforts to improve targeting specificity of a sgRNA.</description><identifier>ISSN: 2045-2322</identifier><identifier>EISSN: 2045-2322</identifier><identifier>DOI: 10.1038/srep40638</identifier><identifier>PMID: 28098181</identifier><language>eng</language><publisher>London: Nature Publishing Group UK</publisher><subject>42/41 ; 631/337 ; 631/61 ; Base Pairing ; Base Sequence ; CRISPR ; CRISPR-Cas Systems ; Endonuclease ; Gene Expression Profiling ; Genes, Reporter ; Genetic Engineering ; Genome ; Genomes ; Humanities and Social Sciences ; Humans ; multidisciplinary ; Mutation ; Nucleotide sequence ; Plasmids ; Ribonucleic acid ; RNA ; RNA, Guide, CRISPR-Cas Systems - chemistry ; RNA, Guide, CRISPR-Cas Systems - genetics ; Science ; Science (multidisciplinary)</subject><ispartof>Scientific reports, 2017-01, Vol.7 (1), p.40638-40638, Article 40638</ispartof><rights>The Author(s) 2017</rights><rights>Copyright Nature Publishing Group Jan 2017</rights><rights>Copyright © 2017, The Author(s) 2017 The Author(s)</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c504t-acdc1d7242d86517d8f36f050dadfe8221e669ebfea015be4b74868557e9ba5f3</citedby><cites>FETCH-LOGICAL-c504t-acdc1d7242d86517d8f36f050dadfe8221e669ebfea015be4b74868557e9ba5f3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5241822/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5241822/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,27901,27902,41096,42165,51551,53766,53768</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/28098181$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Zheng, Ting</creatorcontrib><creatorcontrib>Hou, Yingzi</creatorcontrib><creatorcontrib>Zhang, Pingjing</creatorcontrib><creatorcontrib>Zhang, Zhenxi</creatorcontrib><creatorcontrib>Xu, Ying</creatorcontrib><creatorcontrib>Zhang, Letian</creatorcontrib><creatorcontrib>Niu, Leilei</creatorcontrib><creatorcontrib>Yang, Yi</creatorcontrib><creatorcontrib>Liang, Da</creatorcontrib><creatorcontrib>Yi, Fan</creatorcontrib><creatorcontrib>Peng, Wei</creatorcontrib><creatorcontrib>Feng, Wenjian</creatorcontrib><creatorcontrib>Yang, Ying</creatorcontrib><creatorcontrib>Chen, Jianxin</creatorcontrib><creatorcontrib>Zhu, York Yuanyuan</creatorcontrib><creatorcontrib>Zhang, Li-He</creatorcontrib><creatorcontrib>Du, Quan</creatorcontrib><title>Profiling single-guide RNA specificity reveals a mismatch sensitive core sequence</title><title>Scientific reports</title><addtitle>Sci Rep</addtitle><addtitle>Sci Rep</addtitle><description>Targeting specificity is an essential issue in the development of CRISPR-Cas technology. Using a luciferase activation assay, off-target cleavage activity of sgRNA was systematically investigated on single nucleotide-mismatched targets. In addition to confirming that PAM-proximal mismatches are less tolerated than PAM-distal mismatches, our study further identified a “core” sequence that is highly sensitive to target-mismatch. This sequence is of 4-nucleotide long, located at +4 to +7 position upstream of PAM, and positioned in a steric restriction region when assembled into Cas9 endonuclease. Our study also found that, single or multiple target mismatches at this region abolished off-target cleavage mediated by active sgRNAs, thus proposing a principle for gene-specific sgRNA design. 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Using a luciferase activation assay, off-target cleavage activity of sgRNA was systematically investigated on single nucleotide-mismatched targets. In addition to confirming that PAM-proximal mismatches are less tolerated than PAM-distal mismatches, our study further identified a “core” sequence that is highly sensitive to target-mismatch. This sequence is of 4-nucleotide long, located at +4 to +7 position upstream of PAM, and positioned in a steric restriction region when assembled into Cas9 endonuclease. Our study also found that, single or multiple target mismatches at this region abolished off-target cleavage mediated by active sgRNAs, thus proposing a principle for gene-specific sgRNA design. Characterization of a mismatch sensitive “core” sequence not only enhances our understanding of how this elegant system functions, but also facilitates our efforts to improve targeting specificity of a sgRNA.</abstract><cop>London</cop><pub>Nature Publishing Group UK</pub><pmid>28098181</pmid><doi>10.1038/srep40638</doi><tpages>1</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | 42/41 631/337 631/61 Base Pairing Base Sequence CRISPR CRISPR-Cas Systems Endonuclease Gene Expression Profiling Genes, Reporter Genetic Engineering Genome Genomes Humanities and Social Sciences Humans multidisciplinary Mutation Nucleotide sequence Plasmids Ribonucleic acid RNA RNA, Guide, CRISPR-Cas Systems - chemistry RNA, Guide, CRISPR-Cas Systems - genetics Science Science (multidisciplinary) |
| title | Profiling single-guide RNA specificity reveals a mismatch sensitive core sequence |
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