Toward establishing an efficient and versatile gene targeting system in higher plants

Abstract

Abstract Precise modification of plant genomes provides powerful tools for understanding gene function. Furthermore, pinpoint modification of endogenous plant genes is also an attractive method in molecular plant breeding. One of the best methods currently available to induce specific DNA sequence changes into genomes is gene targeting (GT) via homologous recombination (HR). GT can induce a variety of mutations, including substitution of several nucleotides as well as insertions and deletions. Of the various approaches taken to improve GT efficiency, utilization of engineered sequence-specific endonucleases to create targeted DNA double-strand breaks (DSBs) that stimulate HR at breaking sites is one of the most effective. As a universal selection system for GT cells, a target-gene-independent selection system such as positive–negative selection with subsequent elimination of the positive selection marker using piggyBac transposon or HR combined with mega-nuclease allows retention of the desired mutation without any trace of additional exogenous nucleotides. Furthermore, a novel GT system named in planta GT seems to have effectively overcome problems due to low transformation efficiency of the HR template into plant nuclei, and can adjust the timing of DSB induction at the target locus. In the in planta system, an HR template flanked by a nuclease recognition site is stably integrated into the plant genome; subsequent expression of nuclease simultaneously releases the HR template from the genome and induces a DSB at the target site. Here, we review recent developments in GT, giving examples of new techniques improving the efficiency of GT in plants.