N. Gupta, A. Sayer, M. Prater, C. Mastrokalou, K. Saeed, C. Company, C. Hart, R. Miragaia, A. Trehan, Functional Genomics Centre, U. McDermott, M. E. Strauss, D. Ross-Thriepland, D. Walter, A. Kalinka
Pooled CRISPR screens coupled with single-cell RNA sequencing enable high-throughput functional interrogation of gene regulatory networks, yet systematic comparisons of CRISPR knockout (CRISPRko) and CRISPR interference (CRISPRi) remain limited. We established a single-cell CRISPRko workflow and benchmarked it against CRISPRi using 87 sgRNAs targeting 29 unfolded protein response genes. CRISPRko generated transcriptional phenotypes are highly concordant with CRISPRi, and induced comparable pathway-level responses. While CRISPRi allows direct assessment of target gene repression, CRISPRko provides an effective complementary approach for complete loss-of-function studies, expanding the toolkit for single-cell functional genomics.