Wolabu Tezera W, Cong Lili, Park Jong-Jin, Bao Qinyan, Chen Miao, Sun Juan, Xu Bin, Ge Yaxin, Chai Maofeng, Liu Zhipeng, Wang Zeng-Yu
Noble Research Institute, Ardmore, OK, United States.
College of Grassland Science, Qingdao Agricultural University, Qingdao, China.
Front Plant Sci. 2020 Jul 17;11:1063. doi: 10.3389/fpls.2020.01063. eCollection 2020.
Alfalfa () is an outcrossing tetraploid legume species widely cultivated in the world. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system has been successfully used for genome editing in many plant species. However, the use of CRISPR/Cas9 for gene knockout in alfalfa is still very challenging. Our initial single gRNA-CRISPR/Cas9 system had very low mutagenesis efficiency in alfalfa with no mutant phenotype. In order to develop an optimized genome editing system in alfalfa, we constructed multiplex gRNA-CRISPR/Cas9 vectors by a polycistronic tRNA-gRNA approach targeting the stay-green () gene. The replacement of CaMV35S promoter by the ubiquitin promoter (AtUBQ10) to drive Cas9 expression in the multiplex gRNA system led to a significant improvement in genome editing efficiency, whereas modification of the gRNA scaffold resulted in lower editing efficiency. The most effective multiplex system exhibited 75% genotypic mutagenesis efficiency, which is 30-fold more efficient than the single gRNA vector. Importantly, phenotypic change was easily observed in the mutants, and the phenotypic mutation efficiency reached 68%. This highly efficient multiplex gRNA-CRISPR/Cas9 genome editing system allowed the generation of homozygous mutants with a complete knockout of the four allelic copies in the T0 generation. This optimized system offers an effective way of testing gene functions and overcomes a major barrier in the utilization of genome editing for alfalfa improvement.
紫花苜蓿（Medicago sativa）是一种异花授粉的四倍体豆科植物，在世界范围内广泛种植。成簇规律间隔短回文重复序列（CRISPR）/CRISPR相关蛋白9（CRISPR/Cas9）系统已成功应用于许多植物物种的基因组编辑。然而，将CRISPR/Cas9用于紫花苜蓿的基因敲除仍然具有很大挑战性。我们最初的单gRNA-CRISPR/Cas9系统在紫花苜蓿中的诱变效率非常低，没有突变表型。为了开发一种优化的紫花苜蓿基因组编辑系统，我们通过多顺反子tRNA-gRNA方法构建了靶向持绿基因（stay-green，SGR）的多重gRNA-CRISPR/Cas9载体。在多重gRNA系统中，用拟南芥泛素启动子（AtUBQ10）取代花椰菜花叶病毒35S启动子（CaMV35S）来驱动Cas9表达，导致基因组编辑效率显著提高，而gRNA支架的修饰则导致编辑效率降低。最有效的多重系统表现出75%的基因型诱变效率，比单gRNA载体效率高30倍。重要的是，在突变体中很容易观察到表型变化，表型突变效率达到68%。这种高效的多重gRNA-CRISPR/Cas9基因组编辑系统能够在T0代产生四个等位基因拷贝完全敲除的纯合突变体。这种优化系统为测试基因功能提供了一种有效方法，并克服了利用基因组编辑改良紫花苜蓿的一个主要障碍。
