Ella J. Eaton, Bryce Wick, Jeremy S. Chacón, Amy J. Wang, Mitchell Kluesner, Jackson T. Barnes, Bibekananda Kar, Minjing Wang, Matthew J. Johnson, Joseph G. Skeate, Beau R. Webber, Branden S. Moriarity
Adenine base editors (ABEs) are a promising yet underutilized tool for inducing protein knockout compared to Cas9 nuclease, owing in part to a lack of user-friendly platforms for reagent design and implementation. Here, we present a comprehensive workflow to achieve high-efficiency gene knockouts with ABE as an alternative to Cas9 nuclease-based approaches. This includes optimized guide RNA (gRNA) design using SpliceR, a web-based application, followed by genomic and functional validation of ABE-mediated knockouts for several target genes. We validated gRNAs for 14 immunologically relevant targets, using both NGG and NG PAM compatible ABE8e-variants in primary immune cells. To facilitate data analysis, we developed MultiEditRbatch, an updated version of MultiEditR as a user-friendly analysis tool with addition of batch mode for high-throughput analysis of Sanger sequencing data. MultiEditRbatch is available as a web-based application and an R package, enabling robust assessment of base editing outcomes.