MIT Libraries homeMIT Libraries logoDSpace@MIT

MIT
View Item 
  • DSpace@MIT Home
  • MIT Open Access Articles
  • MIT Open Access Articles
  • View Item
  • DSpace@MIT Home
  • MIT Open Access Articles
  • MIT Open Access Articles
  • View Item
JavaScript is disabled for your browser. Some features of this site may not work without it.

DNA targeting specificity of RNA-guided Cas9 nucleases

Author(s)
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; ... Show more Show less
Thumbnail
DownloadZhang_DNA targeting.pdf (1.327Mb)
PUBLISHER_POLICY

Publisher Policy

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.

Terms of use
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.
Metadata
Show full item record
Abstract
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.
Date issued
2013-09
URI
http://hdl.handle.net/1721.1/102691
Department
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
Journal
Nature Biotechnology
Publisher
Nature Publishing Group
Citation
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.
Version: Author's final manuscript
ISSN
1087-0156
1546-1696

Collections
  • MIT Open Access Articles

Browse

All of DSpaceCommunities & CollectionsBy Issue DateAuthorsTitlesSubjectsThis CollectionBy Issue DateAuthorsTitlesSubjects

My Account

Login

Statistics

OA StatisticsStatistics by CountryStatistics by Department
MIT Libraries homeMIT Libraries logo

Find us on

Twitter Facebook Instagram YouTube RSS

MIT Libraries navigation

SearchHours & locationsBorrow & requestResearch supportAbout us
PrivacyPermissionsAccessibility
MIT
Massachusetts Institute of Technology
Content created by the MIT Libraries, CC BY-NC unless otherwise noted. Notify us about copyright concerns.