Identification of novel genetic interactions with Recql4 in a genome-wide CRISPR/Cas9 rescue screen.

This study was performed to provide a detailed understanding of the functions and pathways that intersect with the essential DNA replication factor RECQL4. Mutations in RECQL4 cause Rothmund-Thomson Syndrome (RTS), a familial cancer syndrome associated with early onset osteosarcoma in a high proportion of patients. Using murine RECQL4 mutations that closely map to recurrent human RTS mutations, we performed an unbiased genome-wide screen to identify genes that, when deleted, rescued the phenotypes associated with RECQL4 mutation. Overall design: Hoxb8 immortalised R26-CreERT2 Recql4fl/+ (control) and R26-CreERT2 Recql4fl/R347X (mutant) myeloid cells stably expressing Cas9 were infected with the BRIE lentiviral Mouse CRISPR Knockout Pooled Library, selected with puromycin for 4 days, then tamoxifen treated to delete the Recql4 floxed allele and grown for 14 days with samples collected at day 0 (when tamoxifen is added), day 4 and day 9 to identify guides specifically enriched in the Recql4 R347X mutant cells.