Fig. 2: Pooled screen design.

a Domain architecture of DNMT1 and DNMT3A proteins. Domain abbreviations: DMAP1, DNMT1-associated protein motif; PCNA, proliferating cell nuclear antigen motif; RFTS, replication focus targeting sequence; CXXC, zinc-finger binding domain; BAH, bromo-adjacent homology domain; PWWP, Pro-Trp-Trp-Pro domain; ADD, ATRX-DNMT3-DNMT3L domain; MTase, methyltransferase domain. sgRNAs were designed to tile the coding regions of both genes. b Screening procedure. sgRNAs targeting DNMT1 and DNMT3B were synthesized and constructed as a pooled virus. Non-targeting control sgRNAs and sgRNAs targeting common essential genes were included as negative and positive controls, respectively. Cells were transduced at an MOI between 0.3 and 0.5 and grown on puromycin selection for 3 weeks. Cells were collected 3 days post-puromycin selection and on the last day of the screen. Isolation of genomic DNA and NGS were performed to calculate sgRNA enrichment and depletion over time. sgRNA enrichment and depletion was mapped along the coding regions of DNMT1 and DNMT3B to determine any functional domains essential for viability. c AML cell lines chosen for pooled screens. THP-1, OCI-M1, and NOMO-1 cells are wild-type for DNMT3A, whereas OCI-AML2, SIG-M5, and OCI-AML3 contain mutations within the MTase domain of DNMT3A.