DNA targeting specificity of RNA-guided Cas9 nucleases
The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing1, 2, 3, 4, 5, 6, 7, 8, 9, 10. Here, we characterize SpCas9 targeting specificity in human cells to inform the selection of target sites and av...
| Main Authors: | , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | en_US |
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Nature Publishing Group
2016
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| Online Access: | http://hdl.handle.net/1721.1/102691 https://orcid.org/0000-0003-2782-2509 https://orcid.org/0000-0001-7915-1685 https://orcid.org/0000-0002-2639-9879 |
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| author | Hsu, Patrick Scott, David Arthur Weinstein, Joshua Ran, F. Ann Konermann, Silvana M. Agarwala, Vineeta Li, Yinqing Fine, Eli J. Wu, Xavier Shalem, Ophir Cradick, Thomas J. Marraffini, Luciano A. Bao, Gang Zhang, Feng |
| author2 | Massachusetts Institute of Technology. Department of Biological Engineering |
| author_facet | Massachusetts Institute of Technology. Department of Biological Engineering Hsu, Patrick Scott, David Arthur Weinstein, Joshua Ran, F. Ann Konermann, Silvana M. Agarwala, Vineeta Li, Yinqing Fine, Eli J. Wu, Xavier Shalem, Ophir Cradick, Thomas J. Marraffini, Luciano A. Bao, Gang Zhang, Feng |
| author_sort | Hsu, Patrick |
| collection | MIT |
| description | The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing1, 2, 3, 4, 5, 6, 7, 8, 9, 10. Here, we characterize SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects. Our study evaluates >700 guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted genomic off-target loci in 293T and 293FT cells. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that SpCas9-mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification. To facilitate mammalian genome engineering applications, we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses. |
| first_indexed | 2024-09-23T11:01:33Z |
| format | Article |
| id | mit-1721.1/102691 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T11:01:33Z |
| publishDate | 2016 |
| publisher | Nature Publishing Group |
| record_format | dspace |
| spelling | mit-1721.1/1026912022-09-27T16:37:36Z DNA targeting specificity of RNA-guided Cas9 nucleases Hsu, Patrick Scott, David Arthur Weinstein, Joshua Ran, F. Ann Konermann, Silvana M. Agarwala, Vineeta Li, Yinqing Fine, Eli J. Wu, Xavier Shalem, Ophir Cradick, Thomas J. Marraffini, Luciano A. Bao, Gang Zhang, Feng Massachusetts Institute of Technology. Department of Biological Engineering Massachusetts Institute of Technology. Department of Brain and Cognitive Sciences McGovern Institute for Brain Research at MIT Hsu, Patrick Scott, David Arthur Weinstein, Joshua Ran, F. Ann Konermann, Silvana M. Li, Yinqing Shalem, Ophir Zhang, Feng Agarwala, Vineeta The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing1, 2, 3, 4, 5, 6, 7, 8, 9, 10. Here, we characterize SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects. Our study evaluates >700 guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted genomic off-target loci in 293T and 293FT cells. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that SpCas9-mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification. To facilitate mammalian genome engineering applications, we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses. National Institutes of Health (U.S.) ((NIH) grant R01-GM34277) National Institutes of Health (U.S.) ((NIH) grant R01-CA133404) National Institutes of Health (U.S.) (NIH Director’s Pioneer Award (DP1-MH100706)) National Institutes of Health (U.S.) (NIH Nanomedicine Development Center Award (PN2EY018244)) National Institutes of Health (U.S.) (NIH Transformative R01 grant (R01-DK097768)) 2016-05-26T17:46:55Z 2016-05-26T17:46:55Z 2013-09 2013-03 Article http://purl.org/eprint/type/JournalArticle 1087-0156 1546-1696 http://hdl.handle.net/1721.1/102691 Hsu, Patrick D., David A. Scott, Joshua A. Weinstein, et al. "DNA targeting specificity of RNA-guided Cas9 nucleases." Nature Biotechnology 31:9 (2013) p.827-834. https://orcid.org/0000-0003-2782-2509 https://orcid.org/0000-0001-7915-1685 https://orcid.org/0000-0002-2639-9879 en_US http://dx.doi.org/10.1038/nbt.2647 Nature Biotechnology Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. application/pdf Nature Publishing Group PMC |
| spellingShingle | Hsu, Patrick Scott, David Arthur Weinstein, Joshua Ran, F. Ann Konermann, Silvana M. Agarwala, Vineeta Li, Yinqing Fine, Eli J. Wu, Xavier Shalem, Ophir Cradick, Thomas J. Marraffini, Luciano A. Bao, Gang Zhang, Feng DNA targeting specificity of RNA-guided Cas9 nucleases |
| title | DNA targeting specificity of RNA-guided Cas9 nucleases |
| title_full | DNA targeting specificity of RNA-guided Cas9 nucleases |
| title_fullStr | DNA targeting specificity of RNA-guided Cas9 nucleases |
| title_full_unstemmed | DNA targeting specificity of RNA-guided Cas9 nucleases |
| title_short | DNA targeting specificity of RNA-guided Cas9 nucleases |
| title_sort | dna targeting specificity of rna guided cas9 nucleases |
| url | http://hdl.handle.net/1721.1/102691 https://orcid.org/0000-0003-2782-2509 https://orcid.org/0000-0001-7915-1685 https://orcid.org/0000-0002-2639-9879 |
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