Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells
Bacterial type II CRISPR-Cas9 systems have been widely adapted for RNA-guided genome editing and transcription regulation in eukaryotic cells, yet their in vivo target specificity is poorly understood. Here we mapped genome-wide binding sites of a catalytically inactive Cas9 (dCas9) from Streptococc...
| Main Authors: | , , , , , , , , , , , , |
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| Format: | Article |
| Language: | en_US |
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Nature Publishing Group
2015
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| Online Access: | http://hdl.handle.net/1721.1/96524 https://orcid.org/0000-0003-2782-2509 https://orcid.org/0000-0003-1465-1691 https://orcid.org/0000-0001-7915-1685 https://orcid.org/0000-0002-7256-3158 https://orcid.org/0000-0002-0187-6800 https://orcid.org/0000-0002-2639-9879 https://orcid.org/0000-0003-0369-5269 |
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| author | Wu, Xuebing Cheng, Albert W. Chen, Sidi Jaenisch, Rudolf Zhang, Feng Chiu, Anthony Chun-yin Sharp, Phillip A. Scott, David Arthur Hsu, Patrick Trevino, Alexandro E. Kriz, Andrea J. Dadon, Daniel Benjamin Konermann, Silvana M |
| author2 | Massachusetts Institute of Technology. Department of Biology |
| author_facet | Massachusetts Institute of Technology. Department of Biology Wu, Xuebing Cheng, Albert W. Chen, Sidi Jaenisch, Rudolf Zhang, Feng Chiu, Anthony Chun-yin Sharp, Phillip A. Scott, David Arthur Hsu, Patrick Trevino, Alexandro E. Kriz, Andrea J. Dadon, Daniel Benjamin Konermann, Silvana M |
| author_sort | Wu, Xuebing |
| collection | MIT |
| description | Bacterial type II CRISPR-Cas9 systems have been widely adapted for RNA-guided genome editing and transcription regulation in eukaryotic cells, yet their in vivo target specificity is poorly understood. Here we mapped genome-wide binding sites of a catalytically inactive Cas9 (dCas9) from Streptococcus pyogenes loaded with single guide RNAs (sgRNAs) in mouse embryonic stem cells (mESCs). Each of the four sgRNAs we tested targets dCas9 to between tens and thousands of genomic sites, frequently characterized by a 5-nucleotide seed region in the sgRNA and an NGG protospacer adjacent motif (PAM). Chromatin inaccessibility decreases dCas9 binding to other sites with matching seed sequences; thus 70% of off-target sites are associated with genes. Targeted sequencing of 295 dCas9 binding sites in mESCs transfected with catalytically active Cas9 identified only one site mutated above background levels. We propose a two-state model for Cas9 binding and cleavage, in which a seed match triggers binding but extensive pairing with target DNA is required for cleavage. |
| first_indexed | 2024-09-23T15:15:13Z |
| format | Article |
| id | mit-1721.1/96524 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T15:15:13Z |
| publishDate | 2015 |
| publisher | Nature Publishing Group |
| record_format | dspace |
| spelling | mit-1721.1/965242022-09-29T13:39:02Z Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells Wu, Xuebing Cheng, Albert W. Chen, Sidi Jaenisch, Rudolf Zhang, Feng Chiu, Anthony Chun-yin Sharp, Phillip A. Scott, David Arthur Hsu, Patrick Trevino, Alexandro E. Kriz, Andrea J. Dadon, Daniel Benjamin Konermann, Silvana M Massachusetts Institute of Technology. Department of Biology McGovern Institute for Brain Research at MIT Whitehead Institute for Biomedical Research Koch Institute for Integrative Cancer Research at MIT Wu, Xuebing Chiu, Anthony Chun-yin Chen, Sidi Sharp, Phillip A. Scott, David Arthur Hsu, Patrick Trevino, Alexandro E. Konermann, Silvana M. Zhang, Feng Kriz, Andrea J. Dadon, Daniel Benjamin Jaenisch, Rudolf Bacterial type II CRISPR-Cas9 systems have been widely adapted for RNA-guided genome editing and transcription regulation in eukaryotic cells, yet their in vivo target specificity is poorly understood. Here we mapped genome-wide binding sites of a catalytically inactive Cas9 (dCas9) from Streptococcus pyogenes loaded with single guide RNAs (sgRNAs) in mouse embryonic stem cells (mESCs). Each of the four sgRNAs we tested targets dCas9 to between tens and thousands of genomic sites, frequently characterized by a 5-nucleotide seed region in the sgRNA and an NGG protospacer adjacent motif (PAM). Chromatin inaccessibility decreases dCas9 binding to other sites with matching seed sequences; thus 70% of off-target sites are associated with genes. Targeted sequencing of 295 dCas9 binding sites in mESCs transfected with catalytically active Cas9 identified only one site mutated above background levels. We propose a two-state model for Cas9 binding and cleavage, in which a seed match triggers binding but extensive pairing with target DNA is required for cleavage. National Institutes of Health (U.S.) (Grant RO1-GM34277) National Institutes of Health (U.S.) (Grant R01-CA133404) National Cancer Institute (U.S.) (Grant PO1-CA42063) National Cancer Institute (U.S.) (Cancer Center Support (Core) Grant P30-CA14051) National Institutes of Health (U.S.) (Director's Pioneer Award 1DP1-MH100706) Damon Runyon Cancer Research Foundation Kinship Foundation. Searle Scholars Program Simons Foundation 2015-04-10T18:06:29Z 2015-04-10T18:06:29Z 2014-04 2014-02 Article http://purl.org/eprint/type/JournalArticle 1087-0156 1546-1696 http://hdl.handle.net/1721.1/96524 Wu, Xuebing, David A Scott, Andrea J Kriz, Anthony C Chiu, Patrick D Hsu, Daniel B Dadon, Albert W Cheng, et al. “Genome-Wide Binding of the CRISPR Endonuclease Cas9 in Mammalian Cells.” Nature Biotechnology 32, no. 7 (April 20, 2014): 670–676. https://orcid.org/0000-0003-2782-2509 https://orcid.org/0000-0003-1465-1691 https://orcid.org/0000-0001-7915-1685 https://orcid.org/0000-0002-7256-3158 https://orcid.org/0000-0002-0187-6800 https://orcid.org/0000-0002-2639-9879 https://orcid.org/0000-0003-0369-5269 en_US http://dx.doi.org/10.1038/nbt.2889 Nature Biotechnology Creative Commons Attribution-Noncommercial-Share Alike http://creativecommons.org/licenses/by-nc-sa/4.0/ application/pdf Nature Publishing Group PMC |
| spellingShingle | Wu, Xuebing Cheng, Albert W. Chen, Sidi Jaenisch, Rudolf Zhang, Feng Chiu, Anthony Chun-yin Sharp, Phillip A. Scott, David Arthur Hsu, Patrick Trevino, Alexandro E. Kriz, Andrea J. Dadon, Daniel Benjamin Konermann, Silvana M Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells |
| title | Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells |
| title_full | Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells |
| title_fullStr | Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells |
| title_full_unstemmed | Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells |
| title_short | Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells |
| title_sort | genome wide binding of the crispr endonuclease cas9 in mammalian cells |
| url | http://hdl.handle.net/1721.1/96524 https://orcid.org/0000-0003-2782-2509 https://orcid.org/0000-0003-1465-1691 https://orcid.org/0000-0001-7915-1685 https://orcid.org/0000-0002-7256-3158 https://orcid.org/0000-0002-0187-6800 https://orcid.org/0000-0002-2639-9879 https://orcid.org/0000-0003-0369-5269 |
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