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Nat Biotechnol. 2013 Sep;31(9):827-32. doi: 10.1038/nbt.2647. Epub 2013 Jul 21.

DNA targeting specificity of RNA-guided Cas9 nucleases.

Author information

1
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].

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.

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PMID:
23873081
PMCID:
PMC3969858
DOI:
10.1038/nbt.2647
[Indexed for MEDLINE]
Free PMC Article
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