High-throughput analysis of the activities of xCas9, SpCas9-NG and SpCas9 at matched and mismatched target sequences in human cells
The applications of clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing can be limited by a lack of compatible protospacer adjacent motifs (PAMs), insufficient on-target activity and off-target effects. Here, we report an extensive comparison of the PAM-sequence c...
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| Veröffentlicht in: | Nature biomedical engineering 2020-01, Vol.4 (1), p.111-124 |
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| creator | Kim, Hui Kwon Lee, Sungtae Kim, Younggwang Park, Jinman Min, Seonwoo Choi, Jae Woo Huang, Tony P. Yoon, Sungroh Liu, David R. Kim, Hyongbum Henry |
| description | The applications of clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing can be limited by a lack of compatible protospacer adjacent motifs (PAMs), insufficient on-target activity and off-target effects. Here, we report an extensive comparison of the PAM-sequence compatibilities and the on-target and off-target activities of Cas9 from
Streptococcus pyogenes
(SpCas9) and the SpCas9 variants xCas9 and SpCas9-NG (which are known to have broader PAM compatibility than SpCas9) at 26,478 lentivirally integrated target sequences and 78 endogenous target sites in human cells. We found that xCas9 has the lowest tolerance for mismatched target sequences and that SpCas9-NG has the broadest PAM compatibility. We also show, on the basis of newly identified non-NGG PAM sequences, that SpCas9-NG and SpCas9 can edit six previously unedited endogenous sites associated with genetic diseases. Moreover, we provide deep-learning models that predict the activities of xCas9 and SpCas9-NG at the target sequences. The resulting deeper understanding of the activities of xCas9, SpCas9-NG and SpCas9 in human cells should facilitate their use.
A comparison of compatibilities in protospacer adjacent motifs and of on-target and off-target activities of
Streptococcus pyogenes
Cas9 variants at endogenous sites in human cells enables the editing of new genomic sites associated with genetic diseases. |
| doi_str_mv | 10.1038/s41551-019-0505-1 |
| format | Article |
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Streptococcus pyogenes
(SpCas9) and the SpCas9 variants xCas9 and SpCas9-NG (which are known to have broader PAM compatibility than SpCas9) at 26,478 lentivirally integrated target sequences and 78 endogenous target sites in human cells. We found that xCas9 has the lowest tolerance for mismatched target sequences and that SpCas9-NG has the broadest PAM compatibility. We also show, on the basis of newly identified non-NGG PAM sequences, that SpCas9-NG and SpCas9 can edit six previously unedited endogenous sites associated with genetic diseases. Moreover, we provide deep-learning models that predict the activities of xCas9 and SpCas9-NG at the target sequences. The resulting deeper understanding of the activities of xCas9, SpCas9-NG and SpCas9 in human cells should facilitate their use.
A comparison of compatibilities in protospacer adjacent motifs and of on-target and off-target activities of
Streptococcus pyogenes
Cas9 variants at endogenous sites in human cells enables the editing of new genomic sites associated with genetic diseases.</description><identifier>ISSN: 2157-846X</identifier><identifier>EISSN: 2157-846X</identifier><identifier>DOI: 10.1038/s41551-019-0505-1</identifier><identifier>PMID: 31937939</identifier><language>eng</language><publisher>London: Nature Publishing Group UK</publisher><subject>42 ; 45/41 ; 631/1647/1511 ; 631/1647/1513/1967/3196 ; Biomedical and Life Sciences ; Biomedical Engineering/Biotechnology ; Biomedicine ; Compatibility ; CRISPR ; CRISPR-Associated Protein 9 - genetics ; CRISPR-Cas Systems - genetics ; Deep Learning ; Editing ; Gene Editing - methods ; Genetic disorders ; Genetic Vectors - genetics ; Genomes ; HEK293 Cells ; Humans ; Lentivirus - physiology ; Streptococcus pyogenes ; Streptococcus pyogenes - genetics</subject><ispartof>Nature biomedical engineering, 2020-01, Vol.4 (1), p.111-124</ispartof><rights>The Author(s), under exclusive licence to Springer Nature Limited 2020</rights><rights>2020© The Author(s), under exclusive licence to Springer Nature Limited 2020</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c438t-eb8d306836da8354fde174b9371b7ddbb7b3c2459011dacea0ffdbc38f0fe4203</citedby><cites>FETCH-LOGICAL-c438t-eb8d306836da8354fde174b9371b7ddbb7b3c2459011dacea0ffdbc38f0fe4203</cites><orcidid>0000-0002-9943-7557 ; 0000-0002-4693-738X</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1038/s41551-019-0505-1$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1038/s41551-019-0505-1$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>314,776,780,27901,27902,41464,42533,51294</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/31937939$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Kim, Hui Kwon</creatorcontrib><creatorcontrib>Lee, Sungtae</creatorcontrib><creatorcontrib>Kim, Younggwang</creatorcontrib><creatorcontrib>Park, Jinman</creatorcontrib><creatorcontrib>Min, Seonwoo</creatorcontrib><creatorcontrib>Choi, Jae Woo</creatorcontrib><creatorcontrib>Huang, Tony P.</creatorcontrib><creatorcontrib>Yoon, Sungroh</creatorcontrib><creatorcontrib>Liu, David R.</creatorcontrib><creatorcontrib>Kim, Hyongbum Henry</creatorcontrib><title>High-throughput analysis of the activities of xCas9, SpCas9-NG and SpCas9 at matched and mismatched target sequences in human cells</title><title>Nature biomedical engineering</title><addtitle>Nat Biomed Eng</addtitle><addtitle>Nat Biomed Eng</addtitle><description>The applications of clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing can be limited by a lack of compatible protospacer adjacent motifs (PAMs), insufficient on-target activity and off-target effects. Here, we report an extensive comparison of the PAM-sequence compatibilities and the on-target and off-target activities of Cas9 from
Streptococcus pyogenes
(SpCas9) and the SpCas9 variants xCas9 and SpCas9-NG (which are known to have broader PAM compatibility than SpCas9) at 26,478 lentivirally integrated target sequences and 78 endogenous target sites in human cells. We found that xCas9 has the lowest tolerance for mismatched target sequences and that SpCas9-NG has the broadest PAM compatibility. We also show, on the basis of newly identified non-NGG PAM sequences, that SpCas9-NG and SpCas9 can edit six previously unedited endogenous sites associated with genetic diseases. Moreover, we provide deep-learning models that predict the activities of xCas9 and SpCas9-NG at the target sequences. The resulting deeper understanding of the activities of xCas9, SpCas9-NG and SpCas9 in human cells should facilitate their use.
A comparison of compatibilities in protospacer adjacent motifs and of on-target and off-target activities of
Streptococcus pyogenes
Cas9 variants at endogenous sites in human cells enables the editing of new genomic sites associated with genetic diseases.</description><subject>42</subject><subject>45/41</subject><subject>631/1647/1511</subject><subject>631/1647/1513/1967/3196</subject><subject>Biomedical and Life Sciences</subject><subject>Biomedical Engineering/Biotechnology</subject><subject>Biomedicine</subject><subject>Compatibility</subject><subject>CRISPR</subject><subject>CRISPR-Associated Protein 9 - genetics</subject><subject>CRISPR-Cas Systems - genetics</subject><subject>Deep Learning</subject><subject>Editing</subject><subject>Gene Editing - methods</subject><subject>Genetic disorders</subject><subject>Genetic Vectors - genetics</subject><subject>Genomes</subject><subject>HEK293 Cells</subject><subject>Humans</subject><subject>Lentivirus - physiology</subject><subject>Streptococcus pyogenes</subject><subject>Streptococcus pyogenes - genetics</subject><issn>2157-846X</issn><issn>2157-846X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2020</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><recordid>eNp1kT1PwzAQhi0EolXpD2BBllgYMNix8zWiClqkCgZAYrMc22lSNUmxHURn_jhO0wJCYrrz-bnX53sBOCX4imCaXFtGwpAgTFKEQxwicgCGAQljlLDo9fBXPgBja5cYe5KyNA6PwYD6NE5pOgSfs3JRIFeYpl0U69ZBUYvVxpYWNjl0hYZCuvK9dKXeVj4mwqaX8GndRfQw9bjanaBwsBJOFlptq1Vp90cnzEI7aPVbq2vplcoaFm0laij1amVPwFEuVlaPd3EEXu5unyczNH-c3k9u5kgymjiks0RRHCU0UiKhIcuVJjHL_E9IFiuVZXFGZcDCFBOihNQC57nKJE1ynGsWYDoCF73u2jR-Euu4n7GbQNS6aS0PKE3SlEU08Oj5H3TZtMavpqPiOIqI36CnSE9J01hrdM7XpqyE2XCCeWcS703ifvW8M4kT33O2U26zSqvvjr0lHgh6wPqreqHNz9P_q34Bq3ec6w</recordid><startdate>20200101</startdate><enddate>20200101</enddate><creator>Kim, Hui Kwon</creator><creator>Lee, Sungtae</creator><creator>Kim, Younggwang</creator><creator>Park, Jinman</creator><creator>Min, Seonwoo</creator><creator>Choi, Jae Woo</creator><creator>Huang, Tony P.</creator><creator>Yoon, Sungroh</creator><creator>Liu, David R.</creator><creator>Kim, Hyongbum Henry</creator><general>Nature Publishing Group UK</general><general>Nature Publishing Group</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>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>ABJCF</scope><scope>AFKRA</scope><scope>ARAPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>GNUQQ</scope><scope>HCIFZ</scope><scope>L6V</scope><scope>LK8</scope><scope>M7P</scope><scope>M7S</scope><scope>P5Z</scope><scope>P62</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>PTHSS</scope><scope>7X8</scope><orcidid>https://orcid.org/0000-0002-9943-7557</orcidid><orcidid>https://orcid.org/0000-0002-4693-738X</orcidid></search><sort><creationdate>20200101</creationdate><title>High-throughput analysis of the activities of xCas9, SpCas9-NG and SpCas9 at matched and mismatched target sequences in human cells</title><author>Kim, Hui Kwon ; Lee, Sungtae ; Kim, Younggwang ; Park, Jinman ; Min, Seonwoo ; Choi, Jae Woo ; Huang, Tony P. ; Yoon, Sungroh ; Liu, David R. ; Kim, Hyongbum Henry</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c438t-eb8d306836da8354fde174b9371b7ddbb7b3c2459011dacea0ffdbc38f0fe4203</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2020</creationdate><topic>42</topic><topic>45/41</topic><topic>631/1647/1511</topic><topic>631/1647/1513/1967/3196</topic><topic>Biomedical and Life Sciences</topic><topic>Biomedical Engineering/Biotechnology</topic><topic>Biomedicine</topic><topic>Compatibility</topic><topic>CRISPR</topic><topic>CRISPR-Associated Protein 9 - genetics</topic><topic>CRISPR-Cas Systems - genetics</topic><topic>Deep Learning</topic><topic>Editing</topic><topic>Gene Editing - methods</topic><topic>Genetic disorders</topic><topic>Genetic Vectors - genetics</topic><topic>Genomes</topic><topic>HEK293 Cells</topic><topic>Humans</topic><topic>Lentivirus - physiology</topic><topic>Streptococcus pyogenes</topic><topic>Streptococcus pyogenes - genetics</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Kim, Hui Kwon</creatorcontrib><creatorcontrib>Lee, Sungtae</creatorcontrib><creatorcontrib>Kim, Younggwang</creatorcontrib><creatorcontrib>Park, Jinman</creatorcontrib><creatorcontrib>Min, Seonwoo</creatorcontrib><creatorcontrib>Choi, Jae Woo</creatorcontrib><creatorcontrib>Huang, Tony P.</creatorcontrib><creatorcontrib>Yoon, Sungroh</creatorcontrib><creatorcontrib>Liu, David R.</creatorcontrib><creatorcontrib>Kim, Hyongbum Henry</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Technology Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Materials Science & Engineering Collection</collection><collection>ProQuest Central UK/Ireland</collection><collection>Advanced Technologies & Aerospace Collection</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Technology Collection</collection><collection>Natural Science Collection</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>ProQuest Central Student</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Engineering Collection</collection><collection>ProQuest Biological Science Collection</collection><collection>Biological Science Database</collection><collection>Engineering Database</collection><collection>Advanced Technologies & Aerospace Database</collection><collection>ProQuest Advanced Technologies & Aerospace Collection</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central China</collection><collection>Engineering Collection</collection><collection>MEDLINE - Academic</collection><jtitle>Nature biomedical engineering</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Kim, Hui Kwon</au><au>Lee, Sungtae</au><au>Kim, Younggwang</au><au>Park, Jinman</au><au>Min, Seonwoo</au><au>Choi, Jae Woo</au><au>Huang, Tony P.</au><au>Yoon, Sungroh</au><au>Liu, David R.</au><au>Kim, Hyongbum Henry</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>High-throughput analysis of the activities of xCas9, SpCas9-NG and SpCas9 at matched and mismatched target sequences in human cells</atitle><jtitle>Nature biomedical engineering</jtitle><stitle>Nat Biomed Eng</stitle><addtitle>Nat Biomed Eng</addtitle><date>2020-01-01</date><risdate>2020</risdate><volume>4</volume><issue>1</issue><spage>111</spage><epage>124</epage><pages>111-124</pages><issn>2157-846X</issn><eissn>2157-846X</eissn><abstract>The applications of clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing can be limited by a lack of compatible protospacer adjacent motifs (PAMs), insufficient on-target activity and off-target effects. Here, we report an extensive comparison of the PAM-sequence compatibilities and the on-target and off-target activities of Cas9 from
Streptococcus pyogenes
(SpCas9) and the SpCas9 variants xCas9 and SpCas9-NG (which are known to have broader PAM compatibility than SpCas9) at 26,478 lentivirally integrated target sequences and 78 endogenous target sites in human cells. We found that xCas9 has the lowest tolerance for mismatched target sequences and that SpCas9-NG has the broadest PAM compatibility. We also show, on the basis of newly identified non-NGG PAM sequences, that SpCas9-NG and SpCas9 can edit six previously unedited endogenous sites associated with genetic diseases. Moreover, we provide deep-learning models that predict the activities of xCas9 and SpCas9-NG at the target sequences. The resulting deeper understanding of the activities of xCas9, SpCas9-NG and SpCas9 in human cells should facilitate their use.
A comparison of compatibilities in protospacer adjacent motifs and of on-target and off-target activities of
Streptococcus pyogenes
Cas9 variants at endogenous sites in human cells enables the editing of new genomic sites associated with genetic diseases.</abstract><cop>London</cop><pub>Nature Publishing Group UK</pub><pmid>31937939</pmid><doi>10.1038/s41551-019-0505-1</doi><tpages>14</tpages><orcidid>https://orcid.org/0000-0002-9943-7557</orcidid><orcidid>https://orcid.org/0000-0002-4693-738X</orcidid></addata></record> |
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| subjects | 42 45/41 631/1647/1511 631/1647/1513/1967/3196 Biomedical and Life Sciences Biomedical Engineering/Biotechnology Biomedicine Compatibility CRISPR CRISPR-Associated Protein 9 - genetics CRISPR-Cas Systems - genetics Deep Learning Editing Gene Editing - methods Genetic disorders Genetic Vectors - genetics Genomes HEK293 Cells Humans Lentivirus - physiology Streptococcus pyogenes Streptococcus pyogenes - genetics |
| title | High-throughput analysis of the activities of xCas9, SpCas9-NG and SpCas9 at matched and mismatched target sequences in human cells |
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