PDF(Pubmed)
Abstract：
Gene targeting (GT) for precise gene insertion or swap into pre-defined genomic location has been a bottleneck for expedited soybean precision breeding. We report a robust selectable marker-free GT system in soybean, one of the most economically important crops. An efficient Oh H1-8 (Ochrobactrum haywardense H1-8)-mediated embryonic axis transformation method was used for the delivery of CRISPR-Cas9 components and donor template to regenerate T0 plants 6-8 weeks after transformation. This approach generated up to 3.4% targeted insertion of the donor sequence into the target locus in T0 plants, with ∼ 90% mutation rate observed at the genomic target site. The GT was demonstrated in two genomic sites using two different donor DNA templates without the need for a selectable marker within the template. High-resolution Southern-by-Sequencing analysis identified T1 plants with precise targeted insertion and without unintended plasmid DNA. Unlike previous low-frequency GT reports in soybean that involved particle bombardment-mediated delivery and extensive selection, the method described here is fast, efficient, reproducible, does not require a selectable marker within the donor DNA, and generates nonchimeric plants with heritable GT.
摘要：
用于精确基因插入或交换到预定义的基因组位置的基因打靶(GT)一直是加速大豆精确育种的瓶颈。我们报告了大豆中一个强大的无选择标记GT系统，经济上最重要的作物之一。一种有效的OhH1-8（OchrobactrumhaywardenseH1-8）介导的胚轴转化方法用于递送CRISPR-Cas9组件和供体模板，以在转化后6-8周再生T0植物。这种方法在T0植物中产生高达3.4％的供体序列靶向插入到靶基因座中，在基因组靶位点观察到90%的突变率。使用两个不同的供体DNA模板在两个基因组位点中证明了GT,而不需要模板内的选择标记。高分辨率Southern测序分析鉴定了具有精确靶向插入且没有非预期质粒DNA的T1植物。与以前的大豆低频GT报道不同，这些报道涉及粒子轰击介导的递送和广泛的选择，这里描述的方法很快，高效,可重复,不需要供体DNA内的选择标记，并产生具有可遗传GT的非嵌合植物。
