ABSTRACT
We present an approach that combines a Cas9 that simultaneously targets hundreds of epigenetically diverse endogenous genomic sites with high-throughput sequencing technologies to measure Cas9 dynamics and cellular responses at scale. This massive multiplexing of CRISPR is enabled by means of novel multi-target gRNAs (mgRNAs), degenerate gRNAs that direct Cas9 to a pre-determined number of well-mapped sites. mgRNAs uncovered generalizable insights into Cas9 binding and cleavage, discovering rapid post-cleavage Cas9 departure and repair factor loading at PAM-proximal genomic DNA. Moreover, by bypassing confounding effects from gRNA sequence, mgRNAs unveiled that Cas9 binding is enhanced at chromatin-accessible regions, and Cas9 cleavage is more efficient near transcribed regions. Combined with light-mediated activation and deactivation of Cas9 activity, mgRNAs further enabled high-throughput study of the cellular response to double strand breaks with high temporal resolution, discovering the presence, extent (under 2 kb), and kinetics (~ 0.5 hr) of reversible DNA damage-induced chromatin decompaction. Altogether, this work establishes mgRNAs as a generalizable platform for multiplexing CRISPR and advances our understanding of intracellular Cas9 activity and the DNA damage response at endogenous loci.
Competing Interest Statement
Johns Hopkins University has submitted patent applications on previously published methods for Cas9 activation and deactivation that were used in this study.