SSA-mediated selection marker gene activation enhances relative gene targeting efficiency in plants

The precise manipulation of genome sequences through gene targeting (GT) is beneficial; however, the low efficiency of homology-directed repair (HDR) in seed plants has made GT difficult to achieve. Generation of double-strand breaks (DSBs) at the target DNA site of interest represents a promising approach to facilitate HDR-mediated GT in organisms. Despite recent advances, GT remains a significant challenge in seed plants. To address these challenges, we propose that the efficiency of CRISPR/Cas9-mediated GT could be enhanced by the exclusive selection of plants that exhibit high levels of HDR activity. To test this hypothesis, a surrogate screening system was developed, which consists of a nonfunctional split-selection marker gene. In this system, DSBs generated by CRISPR/Cas9 at the linker sequence of the tandem repeat will be repaired via single-strand annealing (SSA), a subtype of HDR, resulting in the achievement of antibiotic resistance in plants. This approach allows for a 2- to 23-fold increase in precise and heritable GT efficiency in Arabidopsis and rice. The results indicate that screening with SSA-mediated surrogate system can enrich cells and plants with high HDR activity as well as DSB activity, thus facilitating the establishment of highly efficient GTs at target loci in these plants.