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Nat Biotechnol DOI:10.1038/nbt.2889

Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells.

Publication TypeJournal Article
Year of Publication2014
AuthorsWu, X, Scott, DA, Kriz, AJ, Chiu, AC, Hsu, PD, Dadon, DB, Cheng, AW, Trevino, AE, Konermann, S, Chen, S, Jaenisch, R, Zhang, F, Sharp, PA
JournalNat Biotechnol
Date Published2014 Jul
KeywordsAnimals, Base Sequence, Binding Sites, Cells, Cultured, CRISPR-Cas Systems, Deoxyribonuclease I, DNA-Binding Proteins, Embryonic Stem Cells, Genome, Mice, Models, Genetic, Molecular Sequence Data, Protein Binding

Bacterial type II CRISPR-Cas9 systems have been widely adapted for RNA-guided genome editing and transcription regulation in eukaryotic cells, yet their in vivo target specificity is poorly understood. Here we mapped genome-wide binding sites of a catalytically inactive Cas9 (dCas9) from Streptococcus pyogenes loaded with single guide RNAs (sgRNAs) in mouse embryonic stem cells (mESCs). Each of the four sgRNAs we tested targets dCas9 to between tens and thousands of genomic sites, frequently characterized by a 5-nucleotide seed region in the sgRNA and an NGG protospacer adjacent motif (PAM). Chromatin inaccessibility decreases dCas9 binding to other sites with matching seed sequences; thus 70% of off-target sites are associated with genes. Targeted sequencing of 295 dCas9 binding sites in mESCs transfected with catalytically active Cas9 identified only one site mutated above background levels. We propose a two-state model for Cas9 binding and cleavage, in which a seed match triggers binding but extensive pairing with target DNA is required for cleavage.


Alternate JournalNat. Biotechnol.
PubMed ID24752079
PubMed Central IDPMC4145672
Grant ListT32 GM007287 / GM / NIGMS NIH HHS / United States
R01-CA133404 / CA / NCI NIH HHS / United States
P01 CA042063 / CA / NCI NIH HHS / United States
P30 CA014051 / CA / NCI NIH HHS / United States
P30-CA14051 / CA / NCI NIH HHS / United States
R37 HD045022 / HD / NICHD NIH HHS / United States
R37 GM034277 / GM / NIGMS 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
P01-CA42063 / CA / NCI NIH HHS / United States
1DP1-MH100706 / DP / NCCDPHP CDC HHS / United States
R01-GM34277 / GM / NIGMS NIH HHS / United States