Efficient Gene Targeting in Maize Using Inducible CRISPR-Cas9 and Marker-free Donor Template  

作　　者：Pierluigi Barone Emily Wu Brian Lenderts Ajith Anand William Gordon-Kamm Sergei Svitashev Sandeep Kumar 

机构地区：[1]Corteva AgriscienceTM,Johnston,IA 50131,USA

出　　处：《Molecular Plant》2020年第8期1219-1227,共9页分子植物（英文版）

摘　　要：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-integratad 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 TO 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.

关 键 词：DONOR TEMPLATE INSERTION 

分 类 号：S513[农业科学—作物学] Q943.2[生物学—植物学]