DNA targeting specificity of RNA-guided Cas9 nucleases

PD Hsu, DA Scott, JA Weinstein, FA Ran… - Nature …, 2013 - nature.com
PD Hsu, DA Scott, JA Weinstein, FA Ran, S Konermann, V Agarwala, Y Li, EJ Fine, X Wu
Nature biotechnology, 2013nature.com
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 …
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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