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

• Published in: Molecular therapy Vol. 28; no. 1; pp. 19 - 28
• Main Authors: Chung, Cheng-Han, Allen, Alexander G., Sullivan, Neil T., Atkins, Andrew, Nonnemacher, Michael R., Wigdahl, Brian, Dampier, Will
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 08.01.2020; Elsevier Limited; American Society of Gene & Cell Therapy
• Subjects: Base Sequence - genetics; bioinformatics; Cell Line, Tumor; Chromatin; Chromatin - genetics; Chromosomes; CIRCLE-seq; Clustered Regularly Interspaced Short Palindromic Repeats - genetics; Complementarity; Computational Biology - methods; Computer applications; CRISPR; CRISPR-Associated Protein 9 - genetics; CRISPR-Cas Systems - genetics; Databases, Genetic; Deoxyribonuclease; Deoxyribonuclease I - genetics; Deoxyribonucleic acid; DNA; DNA - genetics; DNA repair; DNA sequencing; DNase-seq; Efficiency; Gene Editing - methods; Gene expression; Genome, Human; Genomes; GUIDE-seq; HEK293 Cells; High-Throughput Nucleotide Sequencing; Humans; Nucleotide sequence; Original; Ribonucleic acid; RNA; RNA, Guide, CRISPR-Cas Systems - genetics; RNA-Seq; Transcription, Genetic; Transcriptome; CRISPR; CIRCLE-seq; DNase-seq; RNA-seq; bioinformatics; chromatin; GUIDE-seq
• ISSN: 1525-0016; 1525-0024
• DOI: 10.1016/j.ymthe.2019.10.008

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. [Display omitted] Chromatin configurations that occupy CRISPR targeting sites have been shown to modulate CRISPR-mediated cleavage efficiency. Our computational analyses using integrated genomic data and multiple quantification models have revealed a threshold of DNA accessibility required for detectable CRISPR-mediated cleavage events that is less than what is needed for gene expression.