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bioRxiv · 10.1101/2022.01.18.476836

Massively parallel genomic perturbations with multi-target CRISPR reveal new insights on Cas9 activity and DNA damage responses at endogenous sites

Zou, R. S.·Marin-Gonzalez, A.·Liu, Y.·Liu, H. B.·Shen, L.·Dveirin, R.·Luo, J. X. J.·Kalhor, R.·Ha, T.
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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.

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Zou, R. S., Marin-Gonzalez, A., Liu, Y., Liu, H. B., Shen, L., Dveirin, R., Luo, J. X. J., Kalhor, R., Ha, T.. 2022-01-20. Massively parallel genomic perturbations with multi-target CRISPR reveal new insights on Cas9 activity and DNA damage responses at endogenous sites. https://doi.org/10.1101/2022.01.18.476836

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