In eukaryotic cells, the nucleus DNA is folded into high-dimensional structures, which determines spatial interaction between gene regulators and impacts gene expression [1]. High-throughput chromosome confirmation capture (Hi-C), requiring millions of cells, reveals that chromatin 3D structure consists of subunits such as topologically associating domains (TADs) and chromatin loops [2]. Cohesin- and CCCTC-binding factor (CTCF)-dependent loop extrusion brings distant gene regulation elements together into chromatin loops [3]. During cell division, the loop structure is crucial to chromatin compaction for DNA passage fidelity [4]. However, chromatin loop formation can also lead to Topoisomerase II-dependent DNA double-strand breaks (DSBs) [5], which concentrate mainly in active enhancer and gene promoter regions [6]. Unrepaired DSBs are toxic lesions, and a small number of them can induce cell apoptosis [7], while mis-repaired DSBs can lead to structural variants (SVs) like deletions, insertions, inversions, and translocations [8], [9]. So far, to accumulate DSBs in human cells and examine their effects on chromatin structure remains a major challenge.

In normal human cells, DSBs are rapidly repaired via the non-homologous end joining (NHEJ) pathway, which is the primary pathway for repairing DSBs [10], [11], [12]. Ku (Ku70/Ku80) is the initial factor that binds to the DSB ends during NHEJ, and it then recruits other downstream factors such as DNA-PKcs, XRCC4, XLF, PAXX, and LIG4 to repair the damaged ends [12], [13]. It has been revealed that Ku80 (also known as Ku86, X-ray repair cross complementing 5 or XRCC5) forms heterodimers with Ku70 (XRCC6) and plays important roles in various cellular processes, including NHEJ, telomere maintenance, and regulation of gene transcription [14], [15], [16]. To date, there have been no reported cases of Ku-deficiency in humans.

Studies using mouse models in 2000 demonstrated that Ku80 plays a role in preventing chromosomal aberrations and malignant transformation [17]. Subsequently, Li et al. and Myung et al. discovered that disrupting one allele of Ku86 gene in human HCT116 cells (Ku86+/-) resulted in elevated levels of gross chromosomal recombination, increased p53 (TP53) levels, reduced cell proliferation, extremely shortened telomeres, and slight hypersensitivity to ionizing radiation (IR). In contrast, Ku86-/- HCT116 cells exhibited limited cell divisions (8-10) before undergoing apoptosis [18], [19].

Recently, END-seq has been developed to map DSBs in human genome [20]. Due to the limitations of traditional gene knockout and screening methods, it was not possible to obtain sufficient Ku80-/- cells for high-throughput studies. In this article, we used CRISPR-Cas9 based RNP method to generate Ku80-/- HCT116 cells. The knockout efficiency of Ku80 can be as high as 99% in these cells, allowing us to collect enough cells for studying the hotspots of DSBs with END-seq in the human genome and identifying SVs in the context of human chromatin 3D structure. We provided Ku80-/- HCT116 cells as a system to explore genome fragility during DSB-repair deficiency and investigating how this might affect 3D genomic structure.