Abstract
DNA double-strand breaks (DSBs) both pose threats to genome integrity and are commonly used for genome editing applications. Structural features of DSB ends play key roles in determining DNA repair pathway usage and outcomes during genome editing, but the cellular factors involved in these processes are only partially known. Through genome-wide CRISPRi screening, we identify ERCC6L2 as critical for repairing Cas12a-induced staggered DSBs but irrelevant for Cas9-induced blunt DSBs. We show that ERCC6L2 acts as a protection factor for staggered DSBs with either 5′ or 3′ polarity, preventing large deletions and translocations stemming from DNA damage induced by Cas12a, TALENs, or dual Cas9 nicks. Furthermore, ERCC6L2 loss hyper-sensitizes cells to multiple staggered DSBs induced by promiscuous Cas12a activity or etoposide-induced TOP2 trapping. By combining genetics and biochemical reconstitution, we find that ERCC6L2 counteracts MRE11-RAD50-NBS1 (MRN)-mediated resection by binding and melting staggered DNA ends, thereby promoting accurate end joining. Our data reveal a protective role of ERCC6L2 in staggered-end DSB repair, which suggests the molecular underpinnings of pathology in patients with ERCC6L2 mutations and cautions against using overhang-inducing genome editing tools for their treatment.
Data availability
Sequencing data for the genomic screens, short-read, and long-read sequencing are available in the Sequence Read Archive (SRA) as BioProject with the accession number PRJNA1299151. Aligned ChIP-seq data are available in NCBI’s Gene Expression Omnibus with the GEO Series accession number GSE312241. Analyzed datasets for genomic screens, ChIP-seq, and CAST-seq are available in the Supplementary Information and in Supplementary Data 1 and Supplementary Data 2. Source data are provided with this paper.
Code availability
The following pipelines have used code available on GitHub: DrugZ (https://github.com/hart-lab/drugz), CRISPResso2 (https://github.com/pinellolab/CRISPResso2), RIMA2, SummarizeOntDels (https://github.com/cornlab/summarizeOntDeletions), first version of CASTseq analysis (https://github.com/AG-Boerries/CAST-Seq), DISCOVER-seq (MRE11 ChIP-seq) https://github.com/cornlab/blender. Chromosomal representation of gMulti target sites was generated using karyoploteR (version 1.28.0)77. Genomic annotations for Cas12a gMulti were retrieved using the annotatr package (version 1.28.0)78.
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Acknowledgements
We would like to thank Rolf Turk at IDT for generously supplying AsCas12a-Ultra used in the screen. We would also like to thank Anna Bratus-Neuenschwander at the Functional Genomics Centre Zürich for great assistance with Nanopore sequencing. We would like to thank Tabea Quaderer for assistance in experiments. We would also like to thank Chris Carnie for all the discussions and feedback he provided to this project. We would also like to thank Alasdair Russell and the whole Genome Editing facility at the CRUK Cambridge Institute, and Huw Naylor, Heather Zecchini and the whole Microscopy Facility at the CRUK Cambridge Institute for all their support. We thank Kate Dry for editorial assistance. We thank Stefan Braunshier (Cejka lab) for human RPA. J.E.C. and S.P.J. are funded by the European Research Council (ERC) under the European Union’s Horizon 2020 Research and Innovation Programme (no. 855741-DDREAMM-ERC-2019-SyG). J.E.C. is supported by the NOMIS Foundation and the Lotte und Adolf Hotz-Sprenger Stiftung, Swiss State Secretariat for Education, Research and Innovation (SERI) and SNSF Project Funding grants (310030_188858; 320030_227979). E.J.A. has received support by an EMBO Postdoctoral Fellowship (ALTF 144-2021). J.F. is a recipient of the EMBO Postdoctoral Fellowship (ALTF 220-2021). Research in the SPJ laboratory is supported by Cancer Research UK (CRUK) Discovery Award DRCPGM\100005, CRUK Cambridge Institute core grant SEBINT-2024/100003 and ERC Synergy Award 855741. A.S.B., N.G. and V.G. were supported by ERC Synergy Award 855741; R.B. by CRUK Discovery Award DRCPGM\100005 and a GlaxoSmithKline award to S.P.J.; and A.S.B. by CRUK RadNET Cambridge C17918/A28870 and Wellcome Early Career Award 227014/Z/23/Z. The SPJ laboratory was also supported by core funding grants C6946/A24843 and WT203144 to the Gurdon Institute. P.C. is funded by the Swiss National Science Foundation (SNSF) (Grants 310030_207588 and 310030_205199) and the European Research Council (ERC) (Grant 101018257). T.Ca. was supported by the German Research Foundation (CA 311/4-1 & FANEDIT/EJPRD20-209). G.A. is funded from the German Federal Ministry of Education and Research (BMBF) within the Medical Informatics Funding Scheme EkoEstMed–FKZ 01ZZ2015.
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E.J.A., A.S.B., S.M.S., E.C., R.B., N.G., J.F., and G.C. designed, performed, and analyzed cell-based experiments. A.S.B. and V.G. analyzed cell-based experiments. E.C. and P.C. performed in vitro reconstitution experiments. S.A., G.A., and R.R. performed CAST-seq experiments. M.D.R.G. provided primary BMSCs. T.Ca., P.C., J.E.C., and S.P.J. provided experimental guidance and material support. E.J.A., A.S.B., S.M.S., J.E.C., and S.P.J. wrote the manuscript with contributions from all other authors.
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T.Ca. and G.A. are inventors of CAST-Seq patents (e.g., US11319580B2 and EP3856928B1). J.E.C. is a co-founder and SAB member of Serac Biosciences and an SAB member of Relation Therapeutics, Hornet Bio, and Kano Therapeutics. E.J.A. was partly supported by funding from CSL Behring. S.P.J. works part-time at Insmed Innovation UK Ltd. S.P.J. is a founding partner of Ahren Innovation Capital LLP, a co-founder of Mission Therapeutics Ltd, and is a consultant and shareholder of Genome Therapeutics Ltd. The remaining authors declare no competing interests.
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Aird, E.J., Serrano-Benitez, A., Siegner, S.M. et al. ERCC6L2 ensures repair fidelity for staggered-end DNA double-strand breaks. Nat Commun (2026). https://doi.org/10.1038/s41467-026-69843-w
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DOI: https://doi.org/10.1038/s41467-026-69843-w
