Homo sapiens BAM files from amplicon sequencing pipeline

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. In this study, the DNA repair profiles of over 200 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 and DNA repair profiling in cell lines can be used to anticipate the repair outcomes at a given target site in primary cells. Furthermore, chemical inhibition of DNA-PKcs 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.