On the utility of Cas13d for genetic interaction mapping

Mapping genetic interactions (GIs) is crucial for understanding genetic network complexity. In this study, we investigate the utility of Cas13d, a CRISPR system targeting RNA, for GI mapping and compare it to Cas9 and Cas12a, two DNA nucleases commonly used for GI mapping. We found that Cas13d induced faster target gene perturbation and generated more uniform cell populations with double perturbations than Cas9 or Cas12a. We then encountered Cas13d gRNA-gRNA interference when concatenating gRNAs targeting different genes into one gRNA array, which we overcame by a dual promoter gRNA expression strategy. Moreover, by concatenating three gRNAs targeting the same gene into one array, we were able to maximize the Cas13d-mediated knockdown effects. Combining these strategies enhanced proliferation phenotypes while reducing library size and facilitated reproducible quantification of GIs in oncogenic signaling pathways. Our study highlights the potential of Cas13d for GI mapping, promising advancements in understanding therapeutically-relevant genetic networks.