title: Validation of Synthetic CRISPR Reagents as a Tool for Arrayed Functional Genomic Screening.
abstract: To date, lentiviral-based CRISPR-Cas9 screens have largely been conducted in pooled format. However, numerous assays are not amenable to pooled approaches, and lentiviral screening in arrayed format presents many challenges. We sought to examine synthetic CRISPR reagents in the context of arrayed screening. Experiments were performed using aberrant DNA replication as an assay. Using synthetic CRISPR RNAs targeting the known control gene GMNN in HCT-116 cells stably expressing Cas9, we observed statistically significant phenotype among the majority of transfected cells within 72 hours. Additional studies revealed near complete loss of GMNN protein and editing of GMNN DNA. We next conducted a screen of synthetic CRISPR RNAs directed against 640 ubiquitin-related genes. Screening identified known and novel DNA replication regulators that were also supported by siRNA gene knockdown. Notably, CRISPR screening identified more statistically significant hits than corresponding siRNA screens run in parallel. These results highlight the possibility of using synthetic CRISPR reagents as an arrayed screening tool.
authors.affiliation: Department of Discovery Oncology, Genentech, South San Francisco, CA, United States of America.
authors.forename: Jenille
authors.initials: J
authors.lastname: Tan
authors.affiliation: Department of Discovery Oncology, Genentech, South San Francisco, CA, United States of America.
authors.forename: Scott E
authors.initials: SE
authors.lastname: Martin
mesh_terms: D064113:CRISPR-Cas Systems / Q000235:genetics*; D003110:Colonic Neoplasms / Q000235:genetics* / Q000473:pathology; D004261:DNA Replication / Q000235:genetics*; D064248:Geminin / Q000037:antagonists & inhibitors* / Q000235:genetics; D055785:Gene Knockdown Techniques; D023281:Genomics / Q000379:methods*; D057166:High-Throughput Screening Assays; D006801:Humans; D034741:RNA, Small Interfering / Q000235:genetics; D014407:Tumor Cells, Cultured
chemical_list: C577995:GMNN protein, human; D064248:Geminin; D034741:RNA, Small Interfering