Two Distinct Pathways Support Gene Correction by Single-Stranded Donors at DNA Nicks

Nicks are the most common form of DNA damage. The mechanisms of their repair are fundamental to genomic stability and of practical importance for genome engineering. We define two pathways that support homology-directed repair by single-stranded DNA donors. One depends upon annealing-driven strand synthesis and acts at both nicks and double-strand breaks. The other depends upon annealing-driven heteroduplex correction and acts at nicks. Homology-directed repair via these pathways, as well as mutagenic end joining, are inhibited by RAD51 at nicks but largely independent of RAD51 at double-strand breaks. Guidelines for coordinated design of targets and donors for gene correction emerge from definition of these pathways. This analysis further suggests that naturally occurring nicks may have significant recombinogenic and mutagenic potential that is normally inhibited by RAD51 loading onto DNA, thereby identifying a function for RAD51 in maintenance of genomic stability. [Display omitted] •Two pathways support HDR at nicks by ssDNA donors•Mechanism-based design of donors improves gene correction efficiency•RPA promotes both HDR and mutagenic end joining (mutEJ) at nicks•RAD51 promotes genomic stability at nicks, suppressing both HDR and mutEJ Nicks are the most common form of DNA damage. Davis and Maizels define two distinct pathways that support homology-directed repair (HDR) at nicks and show that conserved factors suppress or promote genomic instability at nicks. This mechanistic understanding defines guidelines for design of efficient donors for gene correction.