Fig. 1: Development and implementation of CRISPRi screens for identifying genes involved in processing cytosine base editing intermediates.

a Schematic of cytosine base editing. Binding of Cas9n to a genomic locus of interest, facilitated by base-pairing between the DNA and the sgRNA, exposes a window of single-stranded DNA to the cytidine deaminase. Cytosines within this window are deaminated to uracil, while the Cas9n cleaves the opposite strand, to produce a U•G intermediate with a nick. This intermediate is processed by the cell to produce either a C•G to T•A outcome (top), or C•G to non-T•A outcomes, the most common of which is a C•G to G•C (bottom). The genetic determinants for the distribution between the C•G to T•A and C•G to G•C outcomes are still unclear. b Schematic of the generation of CRISPRi-expressing K562 cell lines with integrated dox-inducible rA1-SaBE4(ΔUGI). The CRISPRi constructs shown in Supplementary Fig. 2a were transduced into K562 cells, and successfully transduced cells were selected using fluorescence-activated cell sorting (FACS). The resulting CRISPRi-expressing K562 cells were then transfected with the dox-inducible rA1-SaBE4(ΔUGI) construct shown in Supplementary Fig. 2a and a PiggyBac transposase. Cells with rA1-SaBE4(ΔUGI) successfully integrated were selected for with a hygromycin treatment. c Timeline of the C•G to T•A and C•G to G•C screens. For both screens, the cell line described in (b) was transduced with the lentiviral reporter vectors described in Supplementary Fig. 2b at a low multiplicity of infection (MOI < 0.1). Volcano plots from the C•G to T•A (d) and C•G to G•C (e) screens are shown. Representative genes from enriched pathways identified via GOBP analysis are highlighted in colors indicated in the figure legends. Each screen was conducted in n = 2 independent replicates. Gene phenotypes (median log₂ fold change of 5 CRISPRi sgRNAs) and P values were calculated using the MAGeCK RRA. MAGeCK uses two-sided negative binomial tests with FDR correction at the sgRNA level and one-sided permutation-adjusted RRA tests at the gene level. Source data are provided as a Source Data file. Created in BioRender. Gu, S. (2025) https://BioRender.com/b8hi5u8.