RT Journal Article
SR Electronic
T1 Massively parallel genomic perturbations with multi-target CRISPR reveal new insights on Cas9 activity and DNA damage responses at endogenous sites
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2022.01.18.476836
DO 10.1101/2022.01.18.476836
A1 Roger S. Zou
A1 Alberto Marin-Gonzalez
A1 Yang Liu
A1 Hans B. Liu
A1 Leo Shen
A1 Rachel Dveirin
A1 Jay X. J. Luo
A1 Reza Kalhor
A1 Taekjip Ha
YR 2022
UL http://biorxiv.org/content/early/2022/01/20/2022.01.18.476836.abstract
AB 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 StatementJohns Hopkins University has submitted patent applications on previously published methods for Cas9 activation and deactivation that were used in this study.