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PMID: 23873081 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't

DNA targeting specificity of RNA-guided Cas9 nucleases.

Nature biotechnology ·Vol. 31 ·No. 9 ·2013-09-00 ·Pages 827-32

Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X, Shalem O, Cradick TJ, Marraffini LA, Bao G, Zhang F

Abstract

The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing. 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.

MeSH Terms
Bacterial Proteins/genetics Base Pair Mismatch Base Sequence DNA/genetics Deoxyribonucleases/genetics Genetic Engineering/methods Molecular Sequence Data RNA, Guide/genetics Streptococcus pyogenes/genetics Transcription Factors/genetics
Chemicals
Bacterial Proteins RNA, Guide Transcription Factors DNA Deoxyribonucleases
Authors & Affiliations
14 authors, click to expand affiliations / ORCID
Hsu Patrick D
1] Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. [2] McGovern Institute for Brain Research, Department of Brain and Cognitive Sciences, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. [3] Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA. [4].
Scott David A
Weinstein Joshua A
Ran F Ann
Konermann Silvana
Agarwala Vineeta
Li Yinqing
Fine Eli J
Wu Xuebing
Shalem Ophir
Cradick Thomas J
Marraffini Luciano A
Bao Gang
Zhang Feng
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Article Info
Journal
Nature biotechnology
Abbr.
Nat Biotechnol
ISSN
1546-1696
DOI
10.1038/nbt.2647
Published
2013-09-00
Epub
2013-00-21
Pages
827-32
Language
English
Region
United States
NLM ID
9604648
PMCID
PMC3969858
Subset
IM
Grants
NCI NIH HHS · R01-CA133404 · United States
Howard Hughes Medical Institute · United States
NCCDPHP CDC HHS · DP1-MH100706 · United States
NIGMS NIH HHS · T32 GM007753 · United States
NIDDK NIH HHS · R01-DK097768 · United States
NEI NIH HHS · PN2EY018244 · United States
NIGMS NIH HHS · R01 GM034277 · United States
NIDDK NIH HHS · R01 DK097768 · United States
NIGMS NIH HHS · R01-GM34277 · United States
NCI NIH HHS · R01 CA133404 · United States
NIMH NIH HHS · DP1 MH100706 · United States
Corrections
CommentIn
PMID: 24022156
External Links
PubMed source DOI: 10.1038/nbt.2647 PMC Full Text View journal details
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