title: A genome-wide analysis of Cas9 binding specificity using ChIP-seq and targeted sequence capture.
abstract: Clustered regularly interspaced short palindromic repeat (CRISPR) RNA-guided nucleases have gathered considerable excitement as a tool for genome engineering. However, questions remain about the specificity of target site recognition. Cleavage specificity is typically evaluated by low throughput assays (T7 endonuclease I assay, target amplification followed by high-throughput sequencing), which are limited to a subset of potential off-target sites. Here, we used ChIP-seq to examine genome-wide CRISPR binding specificity at gRNA-specific and gRNA-independent sites for two guide RNAs. RNA-guided Cas9 binding was highly specific to the target site while off-target binding occurred at much lower intensities. Cas9-bound regions were highly enriched in NGG sites, a sequence required for target site recognition by Streptococcus pyogenes Cas9. To determine the relationship between Cas9 binding and endonuclease activity, we applied targeted sequence capture, which allowed us to survey 1200 genomic loci simultaneously including potential off-target sites identified by ChIP-seq and by computational prediction. A high frequency of indels was observed at both target sites and one off-target site, while no cleavage activity could be detected at other ChIP-bound regions. Our results confirm the high-specificity of CRISPR endonucleases and demonstrate that sequence capture can be used as a high-throughput genome-wide approach to identify off-target activity.
authors.affiliation: Genome Center and Department of Biochemistry and Molecular Medicine, University of California, Davis, CA 95616, USA.
authors.forename: Henriette
authors.initials: H
authors.lastname: O'Geen
authors.affiliation: Department of Plant Biology and Genome Center, University of California, Davis, CA 95616, USA.
authors.forename: Isabelle M
authors.initials: IM
authors.lastname: Henry
authors.affiliation: Genome Center and Department of Biochemistry and Molecular Medicine, University of California, Davis, CA 95616, USA.
authors.forename: Mital S
authors.initials: MS
authors.lastname: Bhakta
authors.affiliation: Genome Center and Department of Biochemistry and Molecular Medicine, University of California, Davis, CA 95616, USA.
authors.forename: Joshua F
authors.initials: JF
authors.lastname: Meckler
authors.affiliation: Genome Center and Department of Biochemistry and Molecular Medicine, University of California, Davis, CA 95616, USA djsegal@ucdavis.edu.
authors.forename: David J
authors.initials: DJ
authors.lastname: Segal
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D064130:CRISPR-Associated Proteins / Q000235:genetics* / Q000378:metabolism*; D002460:Cell Line; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D055106:Genome-Wide Association Study; D054643:INDEL Mutation; D051379:Mice; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics / Q000378:metabolism; D013297:Streptococcus pyogenes / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D064130:CRISPR-Associated Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems