Abstract SY05-02: Applications of base editor technology in small molecule: Target validation

Abstract

Abstract CRISPR screens that focus on the gene as the unit of information - whether using knockout, interference, or activation - have reinvigorated the field of functional genomics. The recent development of Base Editor technology allows nucleotide-level manipulation, enabling fine-scale investigation of endogenous loci. The low upfront costs of generating a pooled base editing library, coupled with the small scale and relative ease of executing such screens, makes the financial barrier to entry for such experiments low while also not requiring deep experience with screening. One application of this technology is screens that aim to identify resistance mutations in putative targets of small molecules, which provides excellent evidence that the phenotype is caused by on-target activity of the small molecule. This has heretofore typically been an extraordinarily laborious and time-consuming experimental path, by either slowly cultivating spontaneous drug resistant mutants or by generating and screening large scale ORF-variant pools. Base editor screens are well-suited to vastly accelerate this process. Using both A>G and C>T base editors, coupled with variants of the SpCas9 enzyme, we have conducted drug resistance screens with small molecules that target the anti-apoptotic proteins MCL1, BCL2L1, and BCL2, as well as the DNA repair regulator PARP1. These screens identified resistance residues that were also observed in clinical samples, illustrating the utility of this approach. Additionally, base editor screens are excellent tools for studying genetic variants, as we will demonstrate with screens identifying loss-of-function mutations in BRCA1, BRCA2, and TP53. We anticipate that the tools and methodologies described here will facilitate the investigation of impactful variants at a finer and deeper resolution for any locus of interest. Citation Format: Audrey Griffith, Annabel Sangree, Priyanka Roy, Ruth Hanna, John Doench. Applications of base editor technology in small molecule: Target validation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr SY05-02.