Abstract Co-introduction of targeted nucleases and DNA/RNA templates encoding new genomic sequence is the basis for rapid, effective, and iterable gene editing workflows for therapeutic, agricultural, and basic science applications. Extensive optimization of reagent delivery and nuclease activity have improved genome editing workflows, but comparatively few efforts have been made to alter the gene editing activity of template molecules. Here, we report template DNA modified with interstrand crosslinks (ICLs) – xHDRTs - increases editing frequencies in Cas9-directed gene editing workflows by up to five-fold. xHDRTs increase gene editing frequencies independent of DNA template topology, amount of sequence added, or cell type. Gene editing using xHDRTs requires the DNA repair kinase, ATR, and partially requires Fanconi Anemia proteins, including FANCA, but is independent of other ICL-repair pathways. Covalent modification of donor DNA thus presents a compelling opportunity to improve nonviral gene editing workflows.

Interstrand Crosslinks in Donor DNA Boost Gene Editing in Human Cells