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Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq
CRISPR-Cas genome editing induces targeted DNA damage but can also affect off-target sites. Current off-target discovery methods work using purified DNA or specific cellular models but are incapable of direct detection in vivo. We developed DISCOVER-Seq (discovery of in situ Cas off-targets and veri...
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| Published in: | Science (American Association for the Advancement of Science) 2019-04, Vol.364 (6437), p.286-289 |
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| container_end_page | 289 |
| container_issue | 6437 |
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| container_title | Science (American Association for the Advancement of Science) |
| container_volume | 364 |
| creator | Wienert, Beeke Wyman, Stacia K Richardson, Christopher D Yeh, Charles D Akcakaya, Pinar Porritt, Michelle J Morlock, Michaela Vu, Jonathan T Kazane, Katelynn R Watry, Hannah L Judge, Luke M Conklin, Bruce R Maresca, Marcello Corn, Jacob E |
| description | CRISPR-Cas genome editing induces targeted DNA damage but can also affect off-target sites. Current off-target discovery methods work using purified DNA or specific cellular models but are incapable of direct detection in vivo. We developed DISCOVER-Seq (discovery of in situ Cas off-targets and verification by sequencing), a universally applicable approach for unbiased off-target identification that leverages the recruitment of DNA repair factors in cells and organisms. Tracking the precise recruitment of MRE11 uncovers the molecular nature of Cas activity in cells with single-base resolution. DISCOVER-Seq works with multiple guide RNA formats and types of Cas enzymes, allowing characterization of new editing tools. Off-targets can be identified in cell lines and patient-derived induced pluripotent stem cells and during adenoviral editing of mice, paving the way for in situ off-target discovery within individual patient genotypes during therapeutic genome editing. |
| doi_str_mv | 10.1126/science.aav9023 |
| format | article |
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Current off-target discovery methods work using purified DNA or specific cellular models but are incapable of direct detection in vivo. We developed DISCOVER-Seq (discovery of in situ Cas off-targets and verification by sequencing), a universally applicable approach for unbiased off-target identification that leverages the recruitment of DNA repair factors in cells and organisms. Tracking the precise recruitment of MRE11 uncovers the molecular nature of Cas activity in cells with single-base resolution. DISCOVER-Seq works with multiple guide RNA formats and types of Cas enzymes, allowing characterization of new editing tools. Off-targets can be identified in cell lines and patient-derived induced pluripotent stem cells and during adenoviral editing of mice, paving the way for in situ off-target discovery within individual patient genotypes during therapeutic genome editing.</description><identifier>ISSN: 0036-8075</identifier><identifier>EISSN: 1095-9203</identifier><identifier>DOI: 10.1126/science.aav9023</identifier><identifier>PMID: 31000663</identifier><language>eng</language><publisher>United States: The American Association for the Advancement of Science</publisher><subject>Adenoviridae ; Animals ; Cell Line ; Cell lines ; Change detection ; Chromatin Immunoprecipitation ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR ; CRISPR-Associated Protein 9 - chemistry ; CRISPR-Associated Protein 9 - metabolism ; CRISPR-Cas Systems ; Deoxyribonucleic acid ; DNA ; DNA - chemistry ; DNA - genetics ; DNA Breaks, Double-Stranded ; DNA damage ; DNA Repair ; DNA Repair Enzymes - metabolism ; Editing ; Embryos ; Gene Editing - methods ; Gene sequencing ; Genetic modification ; Genome editing ; Genomes ; Genotypes ; Heterogeneity ; Humans ; In vivo methods and tests ; Induced Pluripotent Stem Cells ; K562 Cells ; MRE11 Homologue Protein - genetics ; MRE11 Homologue Protein - metabolism ; MRE11 protein ; Mutation ; Pluripotency ; Proteins ; Recruitment ; Repair ; Ribonucleic acid ; RNA ; RNA, Guide, CRISPR-Cas Systems ; Sequence Analysis, DNA - methods ; Stem cell transplantation ; Stem cells ; Target detection ; Target recognition</subject><ispartof>Science (American Association for the Advancement of Science), 2019-04, Vol.364 (6437), p.286-289</ispartof><rights>Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.</rights><rights>Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c570t-1db0e3599954c84fa906db5e76747a08d8a04bfbf53ac8ece3312017165e26923</citedby><cites>FETCH-LOGICAL-c570t-1db0e3599954c84fa906db5e76747a08d8a04bfbf53ac8ece3312017165e26923</cites><orcidid>0000-0002-2707-6035 ; 0000-0002-5843-9214 ; 0000-0002-4950-7967 ; 0000-0003-3122-4851 ; 0000-0002-7798-5309 ; 0000-0003-1700-0085 ; 0000-0002-8937-8397 ; 0000-0003-3782-5666</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>230,315,786,790,891,2902,2903,27957,27958</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/31000663$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Wienert, Beeke</creatorcontrib><creatorcontrib>Wyman, Stacia K</creatorcontrib><creatorcontrib>Richardson, Christopher D</creatorcontrib><creatorcontrib>Yeh, Charles D</creatorcontrib><creatorcontrib>Akcakaya, Pinar</creatorcontrib><creatorcontrib>Porritt, Michelle J</creatorcontrib><creatorcontrib>Morlock, Michaela</creatorcontrib><creatorcontrib>Vu, Jonathan T</creatorcontrib><creatorcontrib>Kazane, Katelynn R</creatorcontrib><creatorcontrib>Watry, Hannah L</creatorcontrib><creatorcontrib>Judge, Luke M</creatorcontrib><creatorcontrib>Conklin, Bruce R</creatorcontrib><creatorcontrib>Maresca, Marcello</creatorcontrib><creatorcontrib>Corn, Jacob E</creatorcontrib><title>Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq</title><title>Science (American Association for the Advancement of Science)</title><addtitle>Science</addtitle><description>CRISPR-Cas genome editing induces targeted DNA damage but can also affect off-target sites. Current off-target discovery methods work using purified DNA or specific cellular models but are incapable of direct detection in vivo. We developed DISCOVER-Seq (discovery of in situ Cas off-targets and verification by sequencing), a universally applicable approach for unbiased off-target identification that leverages the recruitment of DNA repair factors in cells and organisms. Tracking the precise recruitment of MRE11 uncovers the molecular nature of Cas activity in cells with single-base resolution. DISCOVER-Seq works with multiple guide RNA formats and types of Cas enzymes, allowing characterization of new editing tools. Off-targets can be identified in cell lines and patient-derived induced pluripotent stem cells and during adenoviral editing of mice, paving the way for in situ off-target discovery within individual patient genotypes during therapeutic genome editing.</description><subject>Adenoviridae</subject><subject>Animals</subject><subject>Cell Line</subject><subject>Cell lines</subject><subject>Change detection</subject><subject>Chromatin Immunoprecipitation</subject><subject>Clustered Regularly Interspaced Short Palindromic Repeats</subject><subject>CRISPR</subject><subject>CRISPR-Associated Protein 9 - chemistry</subject><subject>CRISPR-Associated Protein 9 - metabolism</subject><subject>CRISPR-Cas Systems</subject><subject>Deoxyribonucleic acid</subject><subject>DNA</subject><subject>DNA - chemistry</subject><subject>DNA - genetics</subject><subject>DNA Breaks, Double-Stranded</subject><subject>DNA damage</subject><subject>DNA Repair</subject><subject>DNA Repair Enzymes - metabolism</subject><subject>Editing</subject><subject>Embryos</subject><subject>Gene Editing - methods</subject><subject>Gene sequencing</subject><subject>Genetic modification</subject><subject>Genome editing</subject><subject>Genomes</subject><subject>Genotypes</subject><subject>Heterogeneity</subject><subject>Humans</subject><subject>In vivo methods and tests</subject><subject>Induced Pluripotent Stem Cells</subject><subject>K562 Cells</subject><subject>MRE11 Homologue Protein - genetics</subject><subject>MRE11 Homologue Protein - metabolism</subject><subject>MRE11 protein</subject><subject>Mutation</subject><subject>Pluripotency</subject><subject>Proteins</subject><subject>Recruitment</subject><subject>Repair</subject><subject>Ribonucleic acid</subject><subject>RNA</subject><subject>RNA, Guide, CRISPR-Cas Systems</subject><subject>Sequence Analysis, DNA - methods</subject><subject>Stem cell transplantation</subject><subject>Stem cells</subject><subject>Target detection</subject><subject>Target recognition</subject><issn>0036-8075</issn><issn>1095-9203</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2019</creationdate><recordtype>article</recordtype><recordid>eNpdkUFP4zAQhS3ECkqXMzcUiQuXwDiOnfiChEKBSkisWsrVcpxJMWptiJNK_Hu8S6l29zCakeabp3l6hJxQuKA0E5fBWHQGL7TeSMjYHhlRkDyVGbB9MgJgIi2h4IfkKIRXgLiT7IAcMhpnIdiIVAtXWx2wSRrs0fTWu8S3STWbzn_N4tSmve6W2IfEumRjNz4ZgnXL5GY6rx6fJ7N0ju8_yY9WrwIeb_uYLG4nT9V9-vB4N62uH1LDC-hT2tSAjEspeW7KvNUSRFNzLESRFxrKptSQ123dcqZNiQYZoxnQggqOmZAZG5OrL923oV5jY9D1nV6pt86udfehvLbq342zL2rpN0rwUoIUUeB8K9D59wFDr9Y2GFyttEM_BJVllMqciVhjcvYf-uqHzkV7f6hogxdFpC6_KNP5EDpsd89QUL8DUtuA1DageHH6t4cd_50I-wRhDYy3</recordid><startdate>20190419</startdate><enddate>20190419</enddate><creator>Wienert, Beeke</creator><creator>Wyman, Stacia K</creator><creator>Richardson, Christopher D</creator><creator>Yeh, Charles D</creator><creator>Akcakaya, Pinar</creator><creator>Porritt, Michelle J</creator><creator>Morlock, Michaela</creator><creator>Vu, Jonathan T</creator><creator>Kazane, Katelynn R</creator><creator>Watry, Hannah L</creator><creator>Judge, Luke M</creator><creator>Conklin, Bruce R</creator><creator>Maresca, Marcello</creator><creator>Corn, Jacob E</creator><general>The American Association for the Advancement of Science</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QF</scope><scope>7QG</scope><scope>7QL</scope><scope>7QP</scope><scope>7QQ</scope><scope>7QR</scope><scope>7SC</scope><scope>7SE</scope><scope>7SN</scope><scope>7SP</scope><scope>7SR</scope><scope>7SS</scope><scope>7T7</scope><scope>7TA</scope><scope>7TB</scope><scope>7TK</scope><scope>7TM</scope><scope>7U5</scope><scope>7U9</scope><scope>8BQ</scope><scope>8FD</scope><scope>C1K</scope><scope>F28</scope><scope>FR3</scope><scope>H8D</scope><scope>H8G</scope><scope>H94</scope><scope>JG9</scope><scope>JQ2</scope><scope>K9.</scope><scope>KR7</scope><scope>L7M</scope><scope>L~C</scope><scope>L~D</scope><scope>M7N</scope><scope>P64</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope><orcidid>https://orcid.org/0000-0002-2707-6035</orcidid><orcidid>https://orcid.org/0000-0002-5843-9214</orcidid><orcidid>https://orcid.org/0000-0002-4950-7967</orcidid><orcidid>https://orcid.org/0000-0003-3122-4851</orcidid><orcidid>https://orcid.org/0000-0002-7798-5309</orcidid><orcidid>https://orcid.org/0000-0003-1700-0085</orcidid><orcidid>https://orcid.org/0000-0002-8937-8397</orcidid><orcidid>https://orcid.org/0000-0003-3782-5666</orcidid></search><sort><creationdate>20190419</creationdate><title>Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq</title><author>Wienert, Beeke ; Wyman, Stacia K ; Richardson, Christopher D ; Yeh, Charles D ; Akcakaya, Pinar ; Porritt, Michelle J ; Morlock, Michaela ; Vu, Jonathan T ; Kazane, Katelynn R ; Watry, Hannah L ; Judge, Luke M ; Conklin, Bruce R ; Maresca, Marcello ; Corn, Jacob E</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c570t-1db0e3599954c84fa906db5e76747a08d8a04bfbf53ac8ece3312017165e26923</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2019</creationdate><topic>Adenoviridae</topic><topic>Animals</topic><topic>Cell Line</topic><topic>Cell lines</topic><topic>Change detection</topic><topic>Chromatin Immunoprecipitation</topic><topic>Clustered Regularly Interspaced Short Palindromic Repeats</topic><topic>CRISPR</topic><topic>CRISPR-Associated Protein 9 - chemistry</topic><topic>CRISPR-Associated Protein 9 - metabolism</topic><topic>CRISPR-Cas Systems</topic><topic>Deoxyribonucleic acid</topic><topic>DNA</topic><topic>DNA - chemistry</topic><topic>DNA - genetics</topic><topic>DNA Breaks, Double-Stranded</topic><topic>DNA damage</topic><topic>DNA Repair</topic><topic>DNA Repair Enzymes - metabolism</topic><topic>Editing</topic><topic>Embryos</topic><topic>Gene Editing - methods</topic><topic>Gene sequencing</topic><topic>Genetic modification</topic><topic>Genome editing</topic><topic>Genomes</topic><topic>Genotypes</topic><topic>Heterogeneity</topic><topic>Humans</topic><topic>In vivo methods and tests</topic><topic>Induced Pluripotent Stem Cells</topic><topic>K562 Cells</topic><topic>MRE11 Homologue Protein - genetics</topic><topic>MRE11 Homologue Protein - metabolism</topic><topic>MRE11 protein</topic><topic>Mutation</topic><topic>Pluripotency</topic><topic>Proteins</topic><topic>Recruitment</topic><topic>Repair</topic><topic>Ribonucleic acid</topic><topic>RNA</topic><topic>RNA, Guide, CRISPR-Cas Systems</topic><topic>Sequence Analysis, DNA - 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Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Science (American Association for the Advancement of Science)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Wienert, Beeke</au><au>Wyman, Stacia K</au><au>Richardson, Christopher D</au><au>Yeh, Charles D</au><au>Akcakaya, Pinar</au><au>Porritt, Michelle J</au><au>Morlock, Michaela</au><au>Vu, Jonathan T</au><au>Kazane, Katelynn R</au><au>Watry, Hannah L</au><au>Judge, Luke M</au><au>Conklin, Bruce R</au><au>Maresca, Marcello</au><au>Corn, Jacob E</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq</atitle><jtitle>Science (American Association for the Advancement of Science)</jtitle><addtitle>Science</addtitle><date>2019-04-19</date><risdate>2019</risdate><volume>364</volume><issue>6437</issue><spage>286</spage><epage>289</epage><pages>286-289</pages><issn>0036-8075</issn><eissn>1095-9203</eissn><notes>ObjectType-Article-1</notes><notes>SourceType-Scholarly Journals-1</notes><notes>ObjectType-Feature-2</notes><notes>content type line 23</notes><notes>Author contributions: B.W., S.K.W., C.D.R. and J.E.C. conceived and designed the study and experiments. B.W. performed ChIP-Seq, PCRs for amplicon-NGS and Western Blots in cell lines. S.K.W. analyzed ChIP-Seq and amplicon-NGS data and developed BLENDER software. C.D.Y. performed ChIP-qPCR time course experiments. K.R.K. prepared ChIP-Seq libraries. J.V.T. prepared amplicon-NGS libraries. P.A., M.J.P. and M. Morlock executed intravenous tail vein injections, animal terminations and organ collection and performed Western Blots in mouse tissues. L.M.J. and H.L.W. planned and performed iPSC editing experiments. M. Maresca and B.R.C. supervised experiments and provided expertise. J.E.C. supervised the study. B.W., S.K.W. and J.E.C wrote the manuscript with input from all authors.</notes><notes>authors contributed equally</notes><abstract>CRISPR-Cas genome editing induces targeted DNA damage but can also affect off-target sites. Current off-target discovery methods work using purified DNA or specific cellular models but are incapable of direct detection in vivo. We developed DISCOVER-Seq (discovery of in situ Cas off-targets and verification by sequencing), a universally applicable approach for unbiased off-target identification that leverages the recruitment of DNA repair factors in cells and organisms. Tracking the precise recruitment of MRE11 uncovers the molecular nature of Cas activity in cells with single-base resolution. DISCOVER-Seq works with multiple guide RNA formats and types of Cas enzymes, allowing characterization of new editing tools. Off-targets can be identified in cell lines and patient-derived induced pluripotent stem cells and during adenoviral editing of mice, paving the way for in situ off-target discovery within individual patient genotypes during therapeutic genome editing.</abstract><cop>United States</cop><pub>The American Association for the Advancement of Science</pub><pmid>31000663</pmid><doi>10.1126/science.aav9023</doi><tpages>4</tpages><orcidid>https://orcid.org/0000-0002-2707-6035</orcidid><orcidid>https://orcid.org/0000-0002-5843-9214</orcidid><orcidid>https://orcid.org/0000-0002-4950-7967</orcidid><orcidid>https://orcid.org/0000-0003-3122-4851</orcidid><orcidid>https://orcid.org/0000-0002-7798-5309</orcidid><orcidid>https://orcid.org/0000-0003-1700-0085</orcidid><orcidid>https://orcid.org/0000-0002-8937-8397</orcidid><orcidid>https://orcid.org/0000-0003-3782-5666</orcidid><oa>free_for_read</oa></addata></record> |
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| ispartof | Science (American Association for the Advancement of Science), 2019-04, Vol.364 (6437), p.286-289 |
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| source | American Association for the Advancement of Science |
| subjects | Adenoviridae Animals Cell Line Cell lines Change detection Chromatin Immunoprecipitation Clustered Regularly Interspaced Short Palindromic Repeats CRISPR CRISPR-Associated Protein 9 - chemistry CRISPR-Associated Protein 9 - metabolism CRISPR-Cas Systems Deoxyribonucleic acid DNA DNA - chemistry DNA - genetics DNA Breaks, Double-Stranded DNA damage DNA Repair DNA Repair Enzymes - metabolism Editing Embryos Gene Editing - methods Gene sequencing Genetic modification Genome editing Genomes Genotypes Heterogeneity Humans In vivo methods and tests Induced Pluripotent Stem Cells K562 Cells MRE11 Homologue Protein - genetics MRE11 Homologue Protein - metabolism MRE11 protein Mutation Pluripotency Proteins Recruitment Repair Ribonucleic acid RNA RNA, Guide, CRISPR-Cas Systems Sequence Analysis, DNA - methods Stem cell transplantation Stem cells Target detection Target recognition |
| title | Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq |
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