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Nat Biotechnol DOI:10.1038/nbt.2647

DNA targeting specificity of RNA-guided Cas9 nucleases.

Publication TypeJournal Article
Year of Publication2013
AuthorsHsu, PD, Scott, DA, Weinstein, JA, F Ran, A, Konermann, S, Agarwala, V, Li, Y, Fine, EJ, Wu, X, Shalem, O, Cradick, TJ, Marraffini, LA, Bao, G, Zhang, F
JournalNat Biotechnol
Date Published2013 Sep
KeywordsBacterial Proteins, Base Pair Mismatch, Base Sequence, Deoxyribonucleases, DNA, Genetic Engineering, Molecular Sequence Data, RNA, Guide, Streptococcus pyogenes, Transcription Factors

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.


Alternate JournalNat. Biotechnol.
PubMed ID23873081
PubMed Central IDPMC3969858
Grant ListR01-CA133404 / CA / NCI NIH HHS / United States
DP1-MH100706 / DP / NCCDPHP CDC HHS / United States
T32 GM007753 / GM / NIGMS NIH HHS / United States
R01-DK097768 / DK / NIDDK NIH HHS / United States
PN2EY018244 / EY / NEI NIH HHS / United States
R01 CA133404 / CA / NCI NIH HHS / United States
DP1 MH100706 / MH / NIMH NIH HHS / United States
R01 GM034277 / GM / NIGMS NIH HHS / United States
/ / Howard Hughes Medical Institute / United States
R01 DK097768 / DK / NIDDK NIH HHS / United States
R01-GM34277 / GM / NIGMS NIH HHS / United States