Concurrent live imaging of DNA double-strand break repair and cell-cycle progression by CRISPR/Cas9-mediated knock-in of a tricistronic vector

• Published in: Scientific reports Vol. 8; no. 1; pp. 17309 - 9
• Main Authors: Otsuka, Kensuke, Tomita, Masanori
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 23.11.2018; Nature Publishing Group
• Subjects: 13; 13/109; 13/31; 13/44; 14; 14/19; 14/35; 38/22; 631/1647/245/2186; 631/337/1427/2122; 631/61/54/992; 631/80/641/2350; Animals; Cancer; Cell Cycle; Cell Cycle Proteins; Cell division; CRISPR; CRISPR-Cas Systems; Deoxyribonucleic acid; DNA; DNA Breaks, Double-Stranded; DNA damage; DNA probes; DNA Repair; Double-strand break repair; Fluorescent Antibody Technique; Genetic Vectors - administration & dosage; Genome editing; Genomes; Humanities and Social Sciences; Image Processing, Computer-Assisted - methods; Ionizing radiation; Irradiation; Kinetics; Mice; multidisciplinary; NIH 3T3 Cells; p53 Protein; Patched protein; Plasmids; Probes; Radiation, Ionizing; Risk assessment; Science; Science (multidisciplinary); Tumor Suppressor p53-Binding Protein 1 - antagonists & inhibitors; Tumor Suppressor p53-Binding Protein 1 - genetics; Tumor Suppressor p53-Binding Protein 1 - metabolism; Ubiquitination; Yeast; Genome Editing; Ical Focus; 53BP1 Foci; Cell-cycle Status; DNA Double-strand Breaks (DSB)
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-018-35642-7

Cell-cycle progression can be arrested by ionizing radiation-induced DNA double-strand breaks (DSBs). Although DSBs are patched by DSB repair systems, which comprise proteins such as p53-binding protein 1 (53BP1), the relationship between DSB repair progression and cell-cycle status in living cells is unclear. The probe FUCCI (fluorescent ubiquitination-based cell-cycle indicator) was previously developed for visualizing cell-cycle status. Here, we established novel live-imaging probes based on custom-designed plasmids designated “Focicles” harboring a tricistronic compartment encoding distinct fluorescent proteins ligated to the murine 53BP1 foci-forming region (FFR) and two cell-cycle indicators that are known components of FUCCI (hCdt1 and hGmnn). We used CRISPR/Cas9-mediated genome editing to obtain Focicle knock-in cell lines in NIH3T3 cells, which were subject to X-ray irradiation that induced comparable numbers of Focicle and endogenous-53BP1 foci. In addition, the Focicle probes enabled the kinetic analysis of both DSB repair and cell-cycle arrest/progression after irradiation, demonstrating that the Focicle knock-in cells progressed to cell division after DNA damage elimination. These newly developed probes can help to gain a better understanding of the dynamics of DSB repair and cell-cycle control to in turn guide cancer treatment development and cancer-risk assessments.