CRISPR/Cas9 generated DSB clusters mimic complex lesions induced by high-LET radiation and shift repair from c-NHEJ to mutagenic repair pathways.

DNA double-strand break (DSB) clusters are a hallmark of high-linear energy transfer (high-LET) radiation and are associated with pronounced biological effects, including reduced cell survival and elevated genomic instability. Our previous work in Chinese hamster cells, engineered with variably designed clusters of I-SceI recognition sites, integrated at multiple genomic locations, revealed that DSB clusters suppress classical non-homologous end-joining (c-NHEJ) and induce chromosomal translocations that ultimately increase cell lethality. Here, we extend this line of investigation to human cell lines and generate DSB clusters using alternative approaches that do not require prior genetic manipulation of the test cell lines. We employ CRISPR/Cas9-technology to generate DSB clusters of specific design at a selected genomic locus and examine their consequences on locus integrity. We target Exon 3 of the human HPRT (hHPRT) gene and introduce single DSBs or DSB clusters of varying numbers and inter-DSB distances. Alterations at the locus reflecting hHPRT gene inactivation, are quantified as mutations causing resistance to 6-thioguanine (6TG). Our results show that DSB clusters are markedly more potent inducers of mutations than single DSBs and that DSBs spaced within ~ 600 base pairs synergize in mutation induction. Mechanistic analyses using small-molecule inhibitors and engineered gene knockout cell lines reveal that the increased mutagenicity of clustered DSBs is primarily mediated by DNA end resection and PARP1-dependent alternative end-joining (alt-EJ) pathways. These findings reinforce the biological relevance of DSB clusters as a severe form of complex DNA damage and provide mechanistic insights into high-LET radiation-induced increased cell killing and genomic instability.