CAS Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, The Innovative Academy of Seed Design, The Chinese Academy of Sciences, Wuhan, 430074, China.
University of Chinese Academy of Sciences, Beijing, 100049, China.
J Integr Plant Biol. 2021 Aug;63(8):1410-1415. doi: 10.1111/jipb.13101. Epub 2021 Jun 25.
Clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been widely used for precise gene editing in plants. However, simultaneous gene editing of multiple homoeoalleles remains challenging, especially in self-incompatible polyploid plants. Here, we simultaneously introduced targeted mutations in all three homoeoalleles of two genes in the self-incompatible allohexaploid tall fescue, using both CRISPR/Cas9 and LbCas12a (LbCpf1) systems. Loss-of-function mutants of FaPDS exhibited albino leaves, while knockout of FaHSP17.9 resulted in impaired heat resistance in T0 generation of tall fescue. Moreover, these mutations were inheritable. Our findings demonstrate the feasibility of generating loss-of-function mutants in T0 generation polyploid perennial grasses using CRISPR/Cas systems.
成簇规律间隔短回文重复 (CRISPR)/CRISPR 相关蛋白 9 (Cas9) 系统已广泛用于植物的精确基因编辑。然而，多个同源等位基因的同时基因编辑仍然具有挑战性，尤其是在自交不亲和的多倍体植物中。在这里，我们使用 CRISPR/Cas9 和 LbCas12a（LbCpf1）系统，同时在自交不亲和的异源六倍体高羊茅中的两个基因的所有三个同源等位基因中引入靶向突变。FaPDS 的功能丧失突变体表现出白化叶，而 FaHSP17.9 的敲除导致高羊茅 T0 代耐热性受损。此外，这些突变是可遗传的。我们的研究结果表明，使用 CRISPR/Cas 系统在 T0 代多倍体多年生禾本科植物中产生功能丧失突变体是可行的。
