DNA Repair Profiling Reveals Nonrandom Outcomes at Cas9-Mediated Breaks

• Published in: Molecular cell Vol. 63; no. 4; pp. 633 - 646
• Main Authors: van Overbeek, Megan, Capurso, Daniel, Carter, Matthew M., Thompson, Matthew S., Frias, Elizabeth, Russ, Carsten, Reece-Hoyes, John S., Nye, Christopher, Gradia, Scott, Vidal, Bastien, Zheng, Jiashun, Hoffman, Gregory R., Fuller, Christopher K., May, Andrew P.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 18.08.2016
• Subjects: Bacterial Proteins - genetics; Bacterial Proteins - metabolism; CRISPR-Associated Protein 9; CRISPR-Cas Systems; DNA Breaks, Double-Stranded; DNA End-Joining Repair; Endonucleases - genetics; Endonucleases - metabolism; Gene Editing; Gene Expression Profiling - methods; HCT116 Cells; HEK293 Cells; Humans; K562 Cells; RNA Interference; RNA, Guide, CRISPR-Cas Systems - genetics; RNA, Guide, CRISPR-Cas Systems - metabolism; Time Factors; Transfection
• ISSN: 1097-2765; 1097-4164
• DOI: 10.1016/j.molcel.2016.06.037

The repair outcomes at site-specific DNA double-strand breaks (DSBs) generated by the RNA-guided DNA endonuclease Cas9 determine how gene function is altered. Despite the widespread adoption of CRISPR-Cas9 technology to induce DSBs for genome engineering, the resulting repair products have not been examined in depth. Here, the DNA repair profiles of 223 sites in the human genome demonstrate that the pattern of DNA repair following Cas9 cutting at each site is nonrandom and consistent across experimental replicates, cell lines, and reagent delivery methods. Furthermore, the repair outcomes are determined by the protospacer sequence rather than genomic context, indicating that DNA repair profiling in cell lines can be used to anticipate repair outcomes in primary cells. Chemical inhibition of DNA-PK enabled dissection of the DNA repair profiles into contributions from c-NHEJ and MMEJ. Finally, this work elucidates a strategy for using “error-prone” DNA-repair machinery to generate precise edits. [Display omitted] •DNA repair profiles of 223 sites in the human genome reveal nonrandom outcomes•The protospacer sequence, not genomic context, determines Cas9 DSB repair outcomes•Each DNA repair profile is composed of specific contributions from c-NHEJ and MMEJ•DNA repair profiling can be used to anticipate repair outcomes in primary cells van Overbeek, Capurso et al. demonstrate that repair outcomes are nonrandom at S. pyogenes Cas9-mediated DSBs and are determined by the protospacer sequence rather than genomic context. DNA repair profiling reveals specific contributions of c-NHEJ and MMEJ at each site and an approach to generate precise edits without exogenous template.