DNA targeting specificity of RNA-guided Cas9 nucleases
Published Date:Jul 21 2013
Source:Nat Biotechnol. 2013; 31(9):827-832.
Funding:DP1 MH100706/MH/NIMH NIH HHS/United States
DP1-MH100706/DP/NCCDPHP CDC HHS/United States
PN2EY018244/EY/NEI NIH HHS/United States
R01 DK097768/DK/NIDDK NIH HHS/United States
R01-CA133404/CA/NCI NIH HHS/United States
R01-DK097768/DK/NIDDK NIH HHS/United States
R01-GM34277/GM/NIGMS NIH HHS/United States
T32 GM007753/GM/NIGMS NIH HHS/United States
Howard Hughes Medical Institute/United States
Description: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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