Gene targeting through one-armed homology-directed repair

Gene targeting allows precise insertion of DNA sequences into specific loci in the genome. We examined gene-targeted lines generated by traditional two-arm homology-directed repair (HDR) and identified a variety of unexpected genome editing events. Here we present a CRISPR/Cas9-based HDR approach that uses a single homology arm. Our gene targeting system directs a Cas9-gRNA complex to create a double-stranded break distal to the intended insertion site, allowing for more flexibility in choosing Cas9 target sites. This technology greatly accelerates the speed at which gene targeted Arabidopsis lines can be developed.