Mol Plant|使用诱导性CRISPR-Cas9和无标记供体模板在玉米中高效基因靶向

CRISPR-CAS9是产生靶向突变和基因组缺失的强大工具。然而，在CRISPR-CAS9技术在植物育种中得到充分应用之前，精确的基因插入或序列替换仍然是一个主要障碍。在这里，我们报道了高频的、可选择的、无标记的玉米基因组内基因打靶(GT)。热休克诱导的Cas9用于产生靶向双链断裂和从预整合的T-DNA同时动员供体模板。该构建体的设计使得供体模板的释放和随后的DNA修复激活了可选择标记基因在供体位点内的表达。这种方法在T0植物中产生了高达4.7%的供体序列定向插入到目标位点，在供体位点和基因组靶位点分别观察到高达86%的供体模板释放和99%的突变率。

不同于以前的植物内或基因组内同源重组报告，原始嵌合GT植株需要在下一代中进行广泛的后代筛选来鉴定非嵌合GT个体，我们的方法在一代内提供了非嵌合可遗传GT。

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.

版权作品，未经PaperRSS书面授权，严禁转载，违者将被追究法律责任。

PaperRSS，关注生命科学，高校院所科研进展。