Details:

Alternative Title:
Nat Biotechnol
Personal Author:
Hsu, Patrick D. ; Scott, David A. ; Weinstein, Joshua A ; Ran, F. Ann ; Konermann, Silvana ; ... More +
Hsu, Patrick D. ; Scott, David A. ; Weinstein, Joshua A ; Ran, F. Ann ; Konermann, Silvana ; Agarwala, Vineeta ; Li, Yinqing ; Fine, Eli J. ; Wu, Xuebing ; Shalem, Ophir ; Cradick, Thomas J. ; Marraffini, Luciano A. ; Bao, Gang ; Zhang, Feng Less -
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.
Subject:
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Article Bacterial Proteins Base Pair Mismatch Base Sequence Deoxyribonucleases DNA Genetic Engineering Molecular Sequence Data RNA, Guide Streptococcus Pyogenes Transcription Factors
Source:
Nat Biotechnol. 2013; 31(9):827-832.
Document Type:
Journal Article
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 ; ... More +
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 Less -
Collection(s):
CDC Public Access
Main Document Checksum:
[+]
urn:sha256:c1ced8dd69e691aa90d9ece43d05bb7906feef6815ab26a57eec4c150a3d691d
Download URL:
https://stacks.cdc.gov/view/cdc/29899/cdc_29899_DS1.pdf
File Type:

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