131. Chromatin-Dependent Loci Accessibility Affects CRISPR-Cas9 Targeting Efficiency

The CRISPR-Cas9 system from Streptococcus pyogenes has been successfully modified to target specific genomic loci in a wide range of organisms, making it a powerful tool for biotechnology and personalized medicine. However, there are several challenges to be addressed before successful application of CRISPR-Cas9 systems for clinical use. Specifically, significant concerns have been raised over off-target effects that may lead to aberrant cellular function, and the efficiency of gRNAs varies widely between target loci. The factors influencing the targeting efficiency of a particular gRNA design are not fully understood. Although several groups have developed algorithms that attempt to identify highly active gRNAs based on the sequence composition of the gRNA, the predicted scores for gRNA designs do not correlate with observed activity in cells, suggesting that factors other than gRNA sequence are at play. Here, we demonstrate that the underlying genomic context, especially the chromatin state of the target locus influences the cleavage efficiency of CRIPSR-Cas9 in a sequence independent manner. Furthermore, we demonstrate that knowledge of the chromatin state may inform the selection of highly active gRNAs, and reveal the specific chromatin marks associated with CRISPR-Cas9 target accessibility. Finally, we postulate that in silico tools that consider both gRNA sequence and chromatin state of the target locus will have greater predictive power and facilitate the selection of highly active gRNAs for therapeutic applications.