A Universal System of CRISPR/Cas9-Mediated Gene Targeting Using All-in-One Vector in Plants

Homologous recombination-mediated genome editing, also called gene targeting (GT), is an essential technique that allows precise modification of a target sequence, including introduction of point mutations, knock-in of a reporter gene, and/or swapping of a functional domain. However, due to its low frequency, it has been difficult to establish GT approaches that can be applied widely to a large number of plant species. We have developed a simple and universal clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated DNA double-strand break (DSB)-induced GT system using an all-in-one vector comprising a CRISPR/Cas9 expression construct, selectable marker, and GT donor template. This system enabled introduction of targeted point mutations with non-selectable traits into several target genes in both rice and tobacco. Since it was possible to evaluate the GT frequency on endogenous target genes precisely using this system, we investigated the effect of treatment with Rad51-stimulatory compound 1 (RS-1) on the frequency of DSB-induced GT. GT frequency was slightly, but consistently, improved by RS-1 treatment in both target plants.