Fig. 5: Whole intron deletions from cancer genomes provide in vivo evidence of RT-DSBR.

a Schematic of the CRISPR/Cas9 assay to detect a whole intron deletion (WID) in human cells. b Quantification of reads containing precise WIDs (as a fraction of total repair events) at CALR intron 2 in control cells and ones treated with siREV3L (n = 3 biological replicates) with and without a CRISPR/Cas9-mediated DSB. Statistical significance was assessed using an unpaired two-tailed t-tests. Error bars represent the standard error of the mean (± SEM). c Schematic of the bioinformatic pipeline used to analyze deletions in tumors from the MSK-IMPACT database. WIDs were identified as deletions that span a precise entire intron. The blue box highlights a read showing perfect intron loss. d Example of a WID found in the HLA-B gene of a patient sample from the MSK-IMPACT cohort. Read bases that match the reference are displayed in gray, purple “I” represents insertions, and deletions are indicated with a black dash (–). Alignments displayed with light gray borders and white fill have a mapping quality equal to zero, suggesting they may map to multiple regions across the genome. A 245 bp deletion is observed upon targeted NGS that maps precisely to the area corresponding to the intron flanked by Exon 2–3 of the HLA-B gene. e Schematic of the exons spanning the WID in HLA-B with the flanking primers used to confirm the sequence. f Agarose gel depicting the full-length band corresponding to the locus spanning Exon 2–3 in normal MCF-12A cells (N) and the shorted locus with the intron loss in the tumor sample in HLA-B. P₁ represents a patient from the MSK-IMPACT cohort (n = 1 biological replicate). g Sanger sequencing of the PCR products to confirm the presence of the WID in HLA-B. h Graph representing the number of WID observed in the simulated datasets (10,000 MSK-IMPACT-like cohorts). i, j Total number of WIDs over 73,030 total deletions identified in 64,544 tumor samples of the MSK-IMPACT database. The number of expected WIDs was calculated after randomization of the deletion locations across the whole genome. Using two-tailed Fisher’s exact test, empirical p-values were calculated by comparing the observed versus the 10,000 random values (**** p < 0.0001). Schematics in this figure (a, c–e, and h) were created in BioRender. (2025) https://BioRender.com/9tmc1xb. Source data are provided as a Source Data file. See also Supplementary Fig. 6.