Fig. 1: A genome-wide screen implicates ERCC6L2 in the repair of Cas12a-induced damage.

a Workflow for a dropout-based CRISPR inhibition screen to discern hypersensitivities to Cas9 (blunt double-strand break (DSB)) or Cas12a (5’ staggered DSB) editing. The Weissman v2⁶⁶ genome-wide gRNA library contains a modified mCherry (“CasCherry”) engineered with neighboring nuclease target sites to track transduction and editing efficiency. The gRNA cassette is located 2 kb upstream from the nuclease target sites. b Aggregated screen output results comparing gene-level normalized Z (normZ) effect scores of knocked-down genes in Cas12a relative to Cas9 edited populations (Cas12a gRNA counts/Cas9 gRNA counts). Genes selectively depleted in Cas12a edited cells have a positive normZ effect score while genes selectively depleted in Cas9 edited cells have a negative normZ effect score. Notable DNA repair genes are annotated. c Scheme for a fluorescence-based competition assay to validate candidate genes. A dDSB was introduced using the CasCherry targeting gRNA using Cas12a in a 50:50 mixed population of wild-type and ERCC6L2 knockdown (KD) cells. d Competition assay results in K-562 and RPE1 cell types for two different CRISPRi gRNAs targeting ERCC6L2. Lines represent the mean (n = 2 biological replicates). e Scheme for assessing editing outcomes at the genomic level. A single DSB was introduced with a Cas RNP at the 5’ end of TRAC and the surrounding region was PCR amplified with two different primer sets and sequenced using either short-read (Illumina) or long-read (Oxford Nanopore) sequencing technologies. f Editing outcomes binned by indel size based on short-read sequencing (n = 1 biological replicate). NT, non-targeting. g Cumulative read depth based on read length from long-read sequencing (n = 1 biological replicate). Deletions lead to a leftward shift of the curve. The dashed line corresponds to a 1 kb deletion length. h Quantification of the number of reads in long-read sequencing containing greater than a 1 kb deletion from (g). Source data are provided as a Source Data file.