期刊
NUCLEIC ACIDS RESEARCH
卷 49, 期 13, 页码 -出版社
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkab262
关键词
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资金
- National Cancer Institute [R33 CA236670-01A1]
- Emerson Collective Cancer Research Fund
- American Association for Cancer Research AstraZeneca START fellowship [19-40-12-HUSS]
- Cancer Center Support Grant of the National Institutes of Health/National Cancer Institute [P30CA008748]
This study developed a Cas9-based repair system with simplified repair outcomes and converted it into ddPCR readouts for easier use in more laboratories. The experiment found that the key Alt-EJ factor Pol theta only accounts for 50% of total Alt-EJ, SSTR requires BRCA1 and MRE11 activity, and BRCA1 promotes Alt-EJ usage at two-ended DSBs.
Double strand break (DSB) repair primarily occurs through 3 pathways: non-homologous end-joining (NHEJ), alternative end-joining (Alt-EJ), and homologous recombination (HR). Typical methods to measure pathway usage include integrated cassette reporter assays or visualization of DNA damage induced nuclear foci. It is now well understood that repair of Cas9-induced breaks also involves NHEJ, Alt-EJ, and HR pathways, providing a new format to measure pathway usage. Here, we have developed a simple Cas9-based system with validated repair outcomes that accurately represent each pathway and then converted it to a droplet digital PCR (ddPCR) readout, thus obviating the need for Next Generation Sequencing and bioinformatic analysis with the goal to make Cas9-based system accessible to more laboratories. The assay system has reproduced several important insights. First, absence of the key Alt-EJ factor Pol theta only abrogates similar to 50% of total Alt-EJ. Second, single-strand templated repair (SSTR) requires BRCA1 and MRE11 activity, but not BRCA2, establishing that SSTR commonly used in genome editing is not conventional HR. Third, BRCA1 promotes Alt-EJ usage at two-ended DSBs in contrast to BRCA2. This assay can be used in any system, which permits Cas9 delivery and, importantly, allows rapid genotype-to-phenotype correlation in isogenic cell line pairs.
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