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 r...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.展开更多
基金This work was supported by the National Natural Science Foundation of China(31772349,31672482,and 31401915)the Major Science and Technology Innovation Project of Shandong Province(2019JZZY010726).
文摘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.
