The Key Laboratory of Plant Development and Environmental Adaptation Biology, Ministry of Education, School of Life Sciences, Shandong University, Qingdao 266237, China.
Institute of Advanced Agricultural Sciences, Peking University, Weifang 261325, China.
Plant Physiol. 2021 Oct 5;187(2):758-768. doi: 10.1093/plphys/kiab356.
The CRISPR/Cas9 system is a widely used tool for genome editing in plants. In Arabidopsis (Arabidopsis thaliana), egg cell-specific promoters driving Cas9 expression have been applied to reduce the proportion of T1 transformants that are chimeras; however, this approach generally leads to relatively low mutagenesis rates. In this study, a GLABRA2 mutation-based visible selection (GBVS) system was established to enrich nonchimeric mutants among T1 plants generated by an egg cell-specific CRISPR/Cas9 system. GBVS generally enhanced mutation screening, increasing the frequency by 2.58- to 7.50-fold, and 25%-48.15% of T1 plants selected through the GBVS system were homozygous or biallelic mutants, which was 1.71- to 7.86-fold higher than the percentage selected using the original system. The mutant phenotypes of T2 plants were not obviously affected by the glabrous background for all four target genes used in this study. Additionally, the nonchimeric pyrabactin resistance 1 (PYR1)/PYR1-like 1 (PYL1) and PYL2 triple mutant pyr1/pyl1/pyl2 could be obtained in the T1 generation with a ratio of 26.67% when GBVS was applied. Collectively, our results show that compared with the known CRISPR/Cas9 systems, the GBVS system described here saves more time and labor when used for the obtainment of homozygous or biallelic monogenic mutants and nonchimeric polygenic mutants in Arabidopsis.
CRISPR/Cas9 系统是一种广泛应用于植物基因组编辑的工具。在拟南芥（Arabidopsis thaliana）中，已应用卵母细胞特异性启动子驱动 Cas9 表达，以降低 T1 转化体嵌合体的比例；然而，这种方法通常导致相对较低的诱变率。在这项研究中，建立了基于 GLABRA2 突变的可见选择（GBVS）系统，以在卵母细胞特异性 CRISPR/Cas9 系统产生的 T1 植物中富集非嵌合体突变体。GBVS 通常增强了突变筛选，将频率提高了 2.58 到 7.50 倍，通过 GBVS 系统选择的 25%-48.15%的 T1 植物是纯合或双等位突变体，比原始系统选择的比例高 1.71 到 7.86 倍。对于本研究中使用的四个靶基因，T2 代植物的突变表型不受无毛背景的明显影响。此外，当应用 GBVS 时，非嵌合型吡喃菌素抗性 1（PYR1）/PYR1 样 1（PYL1）和 PYL2 三重突变体 pyr1/pyl1/pyl2 可在 T1 代获得，比例为 26.67%。总的来说，与已知的 CRISPR/Cas9 系统相比，当用于获得拟南芥纯合或双等位单基因突变体和非嵌合多基因突变体时，本文描述的 GBVS 系统节省了更多的时间和劳力。
