Convenient,high-efficiency multiplex genome editing in autotetraploid alfalfa using endogenous U6 promoters and visual reporters  

作　　者：Xiuzhi Xia Shihao Li Na Wang Panxu Cheng Butuo Zhu Pengcheng Zhang Dahai Yang Hao Lin Lifang Niu 

机构地区：[1]Biotechnology Research Institute,Chinese Academy of Agricultural Sciences,Beijing 100081,China [2]College of Grassland Science,Shanxi Agricultural University,Jinzhong 030801,China [3]Tobacco Breeding and Biotechnology Research Center,Yunnan Academy of Tobacco Agricultural Sciences,Key Laboratory of Tobacco Biotechnological Breeding,National Tobacco Genetic Engineering Research Center,Kunming 650021,China

出　　处：《aBIOTECH》2025年第1期81-90,共10页生物技术通报(英文版)

基　　金：supported by grants from the Biological Breeding-National Science and Technology Major Project(2022ZD04011,2023ZD04073);National Natural Science Foundation of China(32325035,32071864);Fundamental Research Funds for Central Non-profit Scientific Institution(1610392024008);Central Laboratory of Biotechnology Research Institute,Chinese Academy of Agricultural Sciences,Hainan Seed Industry Laboratory(B23CJ0208);Yunnan Academy of Tobacco Agricultural Sciences(CNTC-YC2022530000241013,CNTC110201501015(JY-02));Hohhot Key R&D Project(2023-JBGS-S-1);Inner Mongolia Center of Pratacultural Technology Innovation(CCPTZX2023B01).

摘　　要：CRISPR/Cas is a simple,robust,versatile tool for plant biology studies and precision plant breeding.However,establishing a high-efficiency gene editing system for multiplex editing of the autotetraploid crop alfalfa(Medicago sativa L.),the most important forage legume worldwide,remains a formidable challenge.Here,we systematically identified endogenous U6 promoters in alfalfa through transient expression via Agrobacterium-mediated infiltration of alfalfa leaves.We further demonstrated the efficacy of the three most active promoters for genome editing using an optimized alfalfa hairy root system.Subsequently,we established an improved CRISPR/Cas9 multiplex system containing three or four tandemly arrayed MsU6-promoter-driven polycistronic tRNA-sgRNA(PTG)expression cassettes,each consisting of three tRNA-sgRNA units,to simultaneously edit three or four alfalfa genes,coupled with the visual reporter RH1 or RUBY.This toolkit showed efficient multiplex editing in the hairy root system with visual selection.We successfully obtained regenerated,red-colored shoots resulting from the stable transformation of alfalfa.These results highlight the potential application of the visual reporter system for the stable transformation of alfalfa.Our improved CRISPR/Cas9 multiplex system enables convenient,high-efficiency multiplex genome editing in alfalfa,providing a versatile toolset to facilitate functional studies of multiple genes and gene families for basic research and the genetic improvement of alfalfa.

关 键 词：ALFALFA Hairy root system MsU6 promoter Multiplex genome editing Visual reporters 

分 类 号：S54[农业科学—作物学]