RRC ID 50506
Author Royba E, Miyamoto T, Natsuko Akutsu S, Hosoba K, Tauchi H, Kudo Y, Tashiro S, Yamamoto T, Matsuura S.
Title Evaluation of ATM heterozygous mutations underlying individual differences in radiosensitivity using genome editing in human cultured cells.
Journal Sci Rep
Abstract Ionizing radiation (IR) induces DNA double-strand breaks (DSBs), which are an initial step towards chromosomal aberrations and cell death. It has been suggested that there are individual differences in radiosensitivity within human populations, and that the variations in DNA repair genes might determine this heterogeneity. However, it is difficult to quantify the effect of genetic variants on the individual differences in radiosensitivity, since confounding factors such as smoking and the diverse genetic backgrounds within human populations affect radiosensitivity. To precisely quantify the effect of a genetic variation on radiosensitivity, we here used the CRISPR-ObLiGaRe (Obligate Ligation-Gated Recombination) method combined with the CRISPR/Cas9 system and a nonhomologous end joining (NHEJ)-mediated knock-in technique in human cultured cells with a uniform genetic background. We generated ATM heterozygous knock-out (ATM +/-) cell clones as a carrier model of a radiation-hypersensitive autosomal-recessive disorder, ataxia-telangiectasia (A-T). Cytokinesis-blocked micronucleus assay and chromosome aberration assay showed that the radiosensitivity of ATM +/- cell clones was significantly higher than that of ATM +/+ cells, suggesting that ATM gene variants are indeed involved in determining individual radiosensitivity. Importantly, the differences in radiosensitivity among the same genotype clones were small, unlike the individual differences in fibroblasts derived from A-T-affected family members.
Volume 7(1)
Pages 5996
Published 2017-7-20
DOI 10.1038/s41598-017-06393-8
PII 10.1038/s41598-017-06393-8
PMID 28729543
PMC PMC5519549
MeSH Ataxia Telangiectasia Mutated Proteins / genetics* Automation CRISPR-Cas Systems / genetics Cells, Cultured Clone Cells Cytokinesis Fibroblasts / metabolism Fibroblasts / pathology Gene Editing* Heterozygote Humans Individuality* Micronucleus Tests Models, Biological Mutation / genetics* Radiation Tolerance / genetics* Recombination, Genetic / genetics
IF 3.998
Times Cited 12
Resource
DNA material pX330-U6-Chimeric_BB-CBh-hSpCas9 sgRNA ATM exon11 (RDB15816)