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DNA targeting specificity of RNA-guided Cas9 nucleases.
|Publication Type||Journal Article|
|Authors||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., and 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.|
|Year of Publication||2013|
|Date Published (YYYY/MM/DD)||2013/07/21|