Massively parallel multi-target CRISPR system interrogates Cas9-basedtarget recognition, DNA cleavage, and DNA repair

Zou Roger S. et al.

CRISPR-Cas9 nucleases, and particularly Streptococcus pyogenes Cas9, are widespread tools for genome editing. However, many aspects of intracellular Cas9 activity and the ensuing DNA damage response remain incompletely characterized. In order to address these issues, we developed a multiplexed CRISPR approach, where a single, degenerate multi-target gRNA (mgRNA) directs the Cas9 enzyme to target hundred endogenous sites at once. When combined with next-generation sequencing readouts, this system enables interrogation of Cas9 activity and DNA double-strand break (DSB) repair response in high-throughput. Here, we present a step-by-step protocol to deliver a Cas9:mgRNA ribonucleoprotein complex into cultured cells and measure key processes related to Cas9 activity and DSB repair.


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September, 2022


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