Minimal activation of the p53 DNA damage response by a modular cytosine base editor enables effective multiplexed gene knockout in induced pluripotent stem cells

Abstract

Precise genome editing of induced pluripotent stem cells (iPSC) holds great promise for engineering advanced cell therapies. CRISPR-Cas systems have been widely adopted in genome engineering applications, however their dependence on genotoxic DNA double strand breaks (DSBs) presents challenges in hypersensitive iPSCs. Base editors are capable of both modifying and ablating gene function without generating DSBs making them an attractive solution for iPSC engineering. Here we report efficient and durable multiplexed target knockout with minimal impact on cell viability and expansion with a cytosine base editor composed of nCas9-UGI and Rat APOBEC1 assembled using the Pin-point platform (nCas9-UGI:rAPO). Minimal p53-mediated DNA damage signalling occurred independently of the number of simultaneous edits installed, and this could be further reduced by modulating the assembly of the base editor complex. Whereas non-homologous end-joining-mediated DNA damage repair led to p53-mediated selection against imprecise editing outcomes and an associated reduction in the on-target efficiency of multiplexed SpCas9 nuclease editing, p53 activity was dispensable for maintaining genome integrity during the base editing process. The Pin-point platform therefore enables the assembly of base editors optimised for high editing efficiency with substantially reduced risk of selecting for defective DNA damage responses inherent to DSB-dependent editing systems.