Using CRISPR to understand DNA break repair
Felipe Cortés
CNIO
CRISPR-based genome editing has enabled systematic loss-of-function studies at unprecedented scale. In pooled CRISPR knockout screens, thousands of genes can be perturbed in parallel, allowing unbiased identification of factors that influence complex cellular processes such as the response to DNA damage. Independently, CRISPR–Cas nucleases provide a programmable source of site-specific DNA double-strand breaks (DSBs) whose repair can be quantitatively interrogated by analyzing the resulting DNA sequence outcomes. Profiling Cas9-induced insertions and deletions (indels) yields molecular information on the mechanisms operating at a break, enabling repair pathways to be distinguished based on their characteristic mutational signatures.
By combining these two properties, we developed REPAIRome, a genome-wide framework that links genetic perturbations to DSB repair outcomes. Using parallel in-pool profiling of Cas9-induced indels across a genome-wide knockout library, REPAIRome uncovers uncharacterized mechanisms, pathways, and factors involved in DSB repair, including opposing roles for XLF and PAXX, a molecular explanation for Cas9-induced multinucleotide insertions, functions of HLTF in Cas9-induced DSB repair, the involvement of the SAGA complex in microhomology-mediated end joining, and an indel mutational signature associated with VHL loss, renal carcinoma, and hypoxia. These results establish REPAIRome as a resource to systematically explore DSB repair mechanisms and their relevance to genome editing and cancer mutational processes.