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

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 tested targets dCas9 to tens to thousands of genomic sites, characterized by a 5-nucleotide seed region in the sgRNA, in addition to an NGG protospacer adjacent motif (PAM). Chromatin inaccessibility prevents dCas9 binding to other sites with matching seed sequences, and consequently 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. We propose a two-state model for Cas9