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Abstract
背景回顾：CRISPR/Cas9 is currently the most powerful tool to generate mutations in plant genomes and more efficient tools are needed as the scale of experiments increases. In the model plant Arabidopsis, the choice of the promoter driving Cas9 expression is critical to generate germline mutations.
提出问题：Several optimal promoters have been reported. However, it is unclear which promoter is ideal as they have not been thoroughly tested side by side. Furthermore, most plant vectors still use one of the two Cas9 nuclear localization sequence (NLS) configurations initially reported.
主要发现：We genotyped more than 6000 Arabidopsis T2 plants to test seven promoters and six types of NLSs across 14 targets to systematically improve the generation of single and multiplex inheritable mutations.
结果：We found that the RPS5A promoter and bipartite NLS were individually the most efficient components. When combined, 99% of T2 plants contained at least one knockout (KO) mutation and 84% contained 4- to 7-plex KOs, the highest multiplexing KO rate in Arabidopsis to date.
结论：These optimizations will be useful to generate higher-order KOs in the germline of Arabidopsis and will likely be applicable to other CRISPR systems as well.
摘 要
CRISPR/Cas9目前是植物基因组编辑的最有力工具，尤其随着现代实验规模扩大更是凸显CRISPR/Cas9工具的高效性。在模式植物拟南芥中，选择驱动Cas9表达的启动子对于创制生殖系突变至关重要。目前，已有一些研究报道了一些可用的启动子。但是，由于并未集中测试，仍不清楚到底哪一个启动子是最合适的。此外，大多数植物载体仍然使用最初报道的两种Cas9核定位序列（NLS）之一。本文中，作者通过对7个启动子和6种NLS在14个靶点上的测试，对超过6000份拟南芥T2代植株的基因分型，系统改良了CRISPR/Cas9系统产生可遗传的单突和多突的能力。作者发现RPS5A启动子和双分型NLS是各自测试试验中为最高效构建。通过将RPS5A启动子和双分型NLS组合在一起，T2代中99%的植株至少包含1个敲除突变，84%的植株包含4～7重的敲除事件，这是目前为止拟南芥中最高重敲除率的记录。本文报道的CRISPR/Cas9系统有助于创制高阶敲除生殖系突变，并且可以用于其他CRISPR系统的改良。
比利时根特大学Thomas B. Jacobs副教授为本文通讯作者，准博士Ward Develtere为本文第一作者。
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