DNA targeting specificity of RNA-guided Cas9 nucleases
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Zhang_DNA targeting.pdf
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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
Date Issued
September 2013
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
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.
MIT 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
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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.
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DOI of Published Version
https://doi.org/10.1038/nbt.2647
