Accelerated drug-resistant variant discovery with an enhanced, scalable mutagenic base editor platform

Personalized cancer therapeutics bring directed treatment options to patients based on their tumor's genetic signature. Unfortunately, tumor genomes are remarkably adaptable, and acquired resistance through gene mutation frequently occurs. Identifying mutations that promote resistance within drug-treated patient populations can be cost, resource, and time intensive. Accordingly, base editing, enabled by Cas9-deaminase domain fusions, has emerged as a promising approach for rapid, large-scale gene variant screening in situ. Here, we adapt and optimize a conditional activation-induced cytidine deaminase (AID)-dead Cas9 (dCas9) system, which demonstrates greater heterogeneity of edits with an expanded footprint compared to the most commonly utilized cytosine base editor, BE4. In combination with a custom single guide RNA (sgRNA) library, we identify individual and compound variants in epidermal growth factor receptor (EGFR) and v-raf murine sarcoma viral oncogene homolog B1 (BRAF) that confer resistance to established EGFR inhibitors. This system and analytical pipeline provide a simple, highly scalable platform for cis or trans drug-modifying variant discovery and for uncovering valuable insights into protein structure-function relationships.