A novel KU70-mutant human leukemic cell line generated using CRISPR-Cas9 shows increased sensitivity to DSB inducing agents and reduced NHEJ activity

• Published in: Biochimica et biophysica acta. General subjects Vol. 1866; no. 12; p. 130246
• Main Authors: Ghosh, Dipayan, Nilavar, Namrata Madan, Raghavan, Sathees C.
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 01.12.2022
• Subjects: cell lines; cell viability; CRISPR-Cas systems; DNA; DNA breaks; DNA repair; DSB repair; etoposide; genes; Genome instability; heterozygosity; humans; irradiation; KU70; mice; mutants; NHEJ; KU70; CRISPR; DSB; Genome instability; DSB repair; IR; NHEJ; DNA breaks
• DOI: 10.1016/j.bbagen.2022.130246
• ISSN: 0304-4165; 1872-8006

KU70 (XRCC6 gene in humans) is one of the proteins in the KU70-KU80 heterodimer which is the first component recruited to broken DNA ends during DNA double-strand break repair through nonhomologous end joining (NHEJ). Previous studies have shown that Ku70 deficient mouse cells are defective in NHEJ and V(D)J recombination. In contrast, heterozygous KU70 mutant human cell lines did not show any significant change in cell viability and sensitivity towards ionizing radiation. In this study, we used CRISPR-Cas9 technique to generate a KU70 mutant (heterozygous) human pre-B leukemic cell line (N6-KU70–2-DG). We observed that the N6-KU70–2-DG cells showed a prominent reduction in the expression of both KU70 mRNA and protein. The mutant cells showed reduced cell viability, increased sensitivity to DSB inducing agents such as ionizing radiation (IR) and etoposide, and increased number of unrepaired DSBs after exposure to IR. In addition, the mutant cells showed a reduction in the NHEJ activity and increased rate of microhomology mediated joining (MMEJ) activity. KU70 mutant cells also revealed enhanced level of senescence markers following irradiation. Thus, we report a novel KU70-mutant leukemic cell line (heterozygous) with reduced NHEJ, which is sensitive to DNA damaging agents, unlike the previously reported other KU heterozygous mutant cell lines. •KU70 (or XRCC6) is one of the first proteins involved in DSB repair by nonhomologous end joining (NHEJ).•We created a novel KU70 mutant human leukemic cell line (NALM6) using CRISPR-Cas9.•These mutant cells showed slower growth and retarded DSB repair kinetics compared to the wild type cells.•In addition, mutant cells showed decreased NHEJ and increased microhomology mediated joining activity.•Upon exposure to ionizing radiation, mutant cells showed a much higher expression of senescence markers.