Computational Analysis Concerning the Impact of DNA Accessibility on CRISPR-Cas9 Cleavage Efficiency.

ABSTRACT

Defining the variables that impact the specificity of CRISPR/Cas9 has been a major research focus. Whereas sequence complementarity between guide RNA and target DNA substantially dictates cleavage efficiency, DNA accessibility of the targeted loci has also been hypothesized to be an important factor. In this study, functional data from two genome-wide assays, genome-wide, unbiased identification of DSBs enabled by sequencing (GUIDE-seq) and circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq), have been computationally analyzed in conjunction with DNA accessibility determined via DNase I-hypersensitive sequencing from the Encyclopedia of DNA Elements (ENCODE) Database and transcriptome from the Sequence Read Archive to determine whether cellular factors influence CRISPR-induced cleavage efficiency. CIRCLE-seq and GUIDE-seq datasets were selected to represent the absence and presence of cellular factors, respectively. Data analysis revealed that correlations between sequence similarity and CRISPR-induced cleavage frequency were altered by the presence of cellular factors that modulated the level of DNA accessibility. The above-mentioned correlation was abolished when cleavage sites were located in less accessible regions. Furthermore, CRISPR-mediated edits were permissive even at regions that were insufficient for most endogenous genes to be expressed. These results provide a strong basis to dissect the contribution of local chromatin modulation markers on CRISPR-induced cleavage efficiency.