Abstract
CRISPR-based genetic screening has revolutionized cancer drug target discovery, yet reliable, multiplex gene editing to reveal synergies between gene targets remains a major challenge. Here, we present a simple and robust CRISPR-Cas12a-based approach for combinatorial genetic screening in cancer cells. By engineering the CRISPR-AsCas12a system with key modifications to the Cas protein and its CRISPR RNA (crRNA), we can achieve high efficiency combinatorial genetic screening. We demonstrate the performance of our optimized AsCas12a (opAsCas12a) through double knockout screening against epigenetic regulators. This screen reveals synthetic sick interactions between Brd9&Jmjd6, Kat6a&Jmjd6, and Brpf1&Jmjd6 in leukemia cells.
Highlights
CRISPR-based genetic screening has revolutionized cancer drug target discovery, yet reliable, multiplex gene editing to reveal synergies between gene targets remains a major challenge
To unleash the intrinsic RNA-processing potential of Cas12a, we considered whether optimization of a Cas12a system could permit its use in combinatorial genetic screening
Given that purified AsCas12a was as active as SpCas[9] in an in vitro editing assay[14], we hypothesized that suboptimal nuclear localization might be partially responsible for the low knockout efficiency of AsCas12a
Summary
CRISPR-based genetic screening has revolutionized cancer drug target discovery, yet reliable, multiplex gene editing to reveal synergies between gene targets remains a major challenge. Unlike Cas[9], Cas12a has intrinsic RNase activity that allows processing of its own crRNA array, enabling multigene editing from a single RNA transcript[13,14] (Supplementary Fig. 1) This established characteristic of Cas12a makes it potentially suited to multiplex editing for combinatorial genetic screening. We optimize the Cas12a system and demonstrate crRNA design principles for this optimized system Together, these modifications achieve sufficient gene editing efficiency in mammalian cells for Cas12a-based dropout genetic screening. These modifications achieve sufficient gene editing efficiency in mammalian cells for Cas12a-based dropout genetic screening We apply this optimized Cas12a system in a double knockout screen to uncover leukemia-specific synthetic sick/lethal genetic interactions
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