TREX1 restricts CRISPR-Cas9 genome editing in human cells

CRISPR-Cas mediated homology-directed repair (HDR) can flexibly introduce desired mutations at targeted sites in a genome. But achieving high HDR efficiencies is a major hurdle in many cellular contexts. Moreover, cells from patients with mutations in DNA repair factors can exhibit low CRISPR-Cas-mediated HDR, complicating genome editing as a potential treatment. We used genome-wide screening in Fanconi anemia (FA) patient lymphoblastic cell lines to uncover suppressors of CRISPR-Cas mediated HDR. Surprisingly, we found that a single exonuclease called TREX1 is an important determinant of HDR efficiency when single-stranded templates are used as a repair template. TREX1 expression acts as a biomarker for CRISPR-Cas mediated HDR, such that cell lines expressing high levels of TREX1 have poor HDR that can be rescued by TREX1 removal. CRISPR-Cas mediated HDR can also be rescued by using single-stranded DNA templates that are chemically protected in a manner consistent with TREX1’s exonucleolytic activity. Overall, our data provide a mechanistic explanation for why some cells are easier to edit than others and suggest a route to increase CRISPR-Cas mediated HDR in TREX-expressing context.