CRISPR-Cas9 is a powerful tool for generating targeted mutations and genomic deletions. However, precise gene insertion or sequence replacement remains a major hurdle before application of CRISPR-Cas9 technology is fully realized in plant breeding. Here, we report high-frequency, selectable marker-free intra-genomic gene targeting (GT) in maize. Heat shock-inducible Cas9 was used for generating targeted double-strand breaks and simultaneous mobilization of the donor template from pre-integrated T-DNA. The construct was designed such that release of the donor template and subsequent DNA repair activated expression of the selectable marker gene within the donor locus. This approach generated up to 4.7% targeted insertion of the donor sequence into the target locus in T0 plants, with up to 86% detected donor template release and 99% mutation rate being observed at the donor loci and the genomic target site, respectively. Unlike previous in planta or intra-genomic homologous recombination reports in which the original chimeric GT plants required extensive progeny screening in the next generation to identify non-chimeric GT individuals, our method provides non-chimeric heritable GT in one generation.

CRISPR-Cas9是生成靶向突变和基因组缺失的强大工具。然而，在植物育种中完全实现CRISPR-Cas9技术的应用之前，精确的基因插入或序列替换仍然是主要障碍。在这里，我们报告了玉米中的高频，无选择标记的基因组内基因靶向（GT）。热激诱导的Cas9用于产生靶向的双链断裂，并同时从预整合的T-DNA中动员供体模板。设计该构建体，使得供体模板的释放和随后的DNA修复激活了供体基因座内选择标记基因的表达。这种方法最多可将T4植物中供体序列定向插入目标位点的4.7％，在供体位点和基因组靶位点分别检测到高达86％的供体模板释放和99％的突变率。不像以前在植物或基因组内同源重组报告中，原始嵌合GT植物需要在下一代中进行广泛的子代筛选以鉴定非嵌合GT个体，我们的方法可以在一代人中提供非嵌合遗传性GT。