Precise and heritable gene targeting in rice using a sequential transformation strategy.

Gene targeting (GT) is a powerful tool for modifying endogenous genomic sequences of interest, such as sequence replacement and gene knockin. Although the efficiency of GT is extremely low in higher plants, engineered sequence-specific nucleases (SSNs)-mediated double-strand breaks (DSBs) can improve GT frequency. We recently reported a CRISPR-Cas9-mediated approach for heritable GT in , called the "sequential transformation" strategy. For efficient establishment of GT via the sequential transformation method, strong Cas9 activity and robust DSBs are required in the plant cells being infected with carrying sgRNA and donor DNA. Accordingly, we generated two independent parental lines with maize promoter-driven Cas9 and established sequential transformation-mediated GT in the Japonica rice cultivar Nipponbare. We achieved precise knockin into the endogenous and loci. We believe that our GT technology could be widely utilized in rice research and breeding applications.