Journal
NATURE BIOTECHNOLOGY
Volume 31, Issue 9, Pages 827-+Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nbt.2647
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Funding
- National Institutes of Health (NIH) [R01-GM34277, R01-CA133404, R01-DK097768]
- NIH Nanomedicine Development Center [PN2EY018244]
- NIH Director's Pioneer Award [DP1-MH100706]
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The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing(1-10). 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.
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