Abstract
The clustered regularly interspaced short palindromic repeat (CRISPR)-associated enzyme Cas9 is an RNA-guided nuclease that has been widely adapted for genome editing in eukaryotic cells. However, the in vivo target specificity of Cas9 is poorly understood and most studies rely on in silico predictions to define the potential off-target editing spectrum. Using chromatin immunoprecipitation followed by sequencing (ChIP-seq), we delineate the genome-wide binding panorama of catalytically inactive Cas9 directed by two different single guide (sg) RNAs targeting the Trp53 locus. Cas9:sgRNA complexes are able to load onto multiple sites with short seed regions adjacent to 5′NGG3′ protospacer adjacent motifs (PAM). Yet among 43 ChIP-seq sites harboring seed regions analyzed for mutational status, we find editing only at the intended on-target locus and one off-target site. In vitro analysis of target site recognition revealed that interactions between the 5′ end of the guide and PAM-distal target sequences are necessary to efficiently engage Cas9 nucleolytic activity, providing an explanation for why off-target editing is significantly lower than expected from ChIP-seq data.
Highlights
The Streptococcus pyogenes clustered regularly interspaced short palindromic repeat (CRISPR) endonuclease Cas9 (CRISPR-associated), in conjunction with a bifunctional single guide RNA that binds Cas9 and targets a,20 nucleotide genomic address via base complementarity, has become the tool of choice for a number of precise genome editing applications ex vivo [1,2,3,4,5,6,7,8] and in vivo [9]
The much shorter motif derived from the sgp53-3/dmCas9 ChIP-seq sample, at odds with the 11–13 nt seed sequence that is generally considered when predicting off-target sites, is in line with recently published ChIPseq data performed with dmCas9 using different sgRNAs [20,21]
Our results help broaden our understanding of the mechanism of Cas9 target specificity
Summary
Two recent ChIP-seq studies have reported that Cas binds a multitude of off-target sites, with some harboring seed sequences as short as 5 nts [20,21]. Virtually no editing activity was detected at a large number of these sites when probed for the presence of mutations, suggesting that target binding per se is insufficient to trigger DNA cleavage [20,21]. In vitro characterization of Cas binding and cleavage revealed variations in efficiencies dictated by interactions between the 59 end of the sgRNA guide region and PAM distal target sequences. Our results indicate that the sequence requirements for Cas DNA binding are different from those for catalytic activity – with PAMdistal target and 59 sgRNA interactions being critical for DNA cleavage
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