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Nature Biotechnology 2013, Vol. 31 (9) :827 -+ doi:10.1038/nbt.2647 <<-上一篇 下一篇 ->>
DNA targeting specificity of RNA-guided Cas9 nucleases
Scott, David A.1,2;Weinstein, Joshua A.1,2;Konermann, Silvana1,2;Li, Yinqing1,2;Shalem, Ophir1,2;Zhang, Feng1,2;Hsu, Patrick D.1,2,3;Ran, F. Ann1,2,3;Agarwala, Vineeta1,4,5;Fine, Eli J.6,7;Cradick, Thomas J.6,7;Bao, Gang6,7;Wu, Xuebing8;Marraffini, Luciano A.9;
Scott, David A.1,2;Weinstein, Joshua A.1,2;Konermann, Silvana1,2;Li, Yinqing1,2;Shalem, Ophir1,2;Zhang, Feng1,2;Hsu, Patrick D.1,2,3;Ran, F. Ann1,2,3;Agarwala, Vineeta1,4,5;Fine, Eli J.6,7;Cradick, Thomas J.6,7;Bao, Gang6,7;Wu, Xuebing8;Marraffini, Luciano A.9;
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(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.
Keywords: MICE SYSTEMS GENERATION ENDONUCLEASE CRISPR GENE GENOME HUMAN-CELLS DOUBLE-STRAND BREAKS
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