Development of a single transcript CRISPR/Cas9 toolkit for efficient genome editing in autotetraploid alfalfa  

作　　者：Haixia Zhao Siyi Zhao Yingping Cao Xiping Jiang Lijuan Zhao Zhimeng Li Mengqi Wang Ruijuan Yang Chuanen Zhou Zhaoming Wang Feng Yuan Dongmei Ma Hao Lin Wenwen Liu Chunxiang Fu 

机构地区：[1]CAS Key Laboratory of Biofuels,Shandong Provincial Key Laboratory of Energy Genetics,Qingdao Institute of Bioenergy and Bioprocess Technology,Chinese Academy of Sciences,Qingdao 266101,Shandong,China [2]Shandong Energy Institute,Qingdao 266101,Shandong,China [3]Qingdao New Energy Shandong Laboratory,Qingdao 266101,Shandong,China [4]University of Chinese Academy of Sciences,Beijing 100049,China [5]The Key Laboratory of Plant Development and Environmental Adaptation Biology,Ministry of Education,School of Life Sciences,Shandong University,Qingdao 266101,Shandong,China [6]Key Laboratory of Forage Breeding and Seed Production of Inner Mongolia,Inner Mongolia M-Grass Ecology and Environment(Group)Co.,Ltd.,Hohhot 010016,Inner Mongolia,China [7]School of Ecology Environment,Ningxia University,Yinchuan 750021,Ningxia,China [8]Biotechnology Research Institute,Chinese Academy of Agricultural Sciences,Beijing 100081,China

出　　处：《The Crop Journal》2024年第3期788-795,共8页作物学报（英文版）

基　　金：supported by the Strategic Priority Research Program of the Chinese Academy of Sciences(XDA26030301);Hohhot Key R&D Project(2023-JBGSS-1),the National Natural Science Foundation of China(U23A200206,32071864,32325035);the Taishan Scholar Program of Shandong(to Chunxiang Fu);the Shandong Provincial Natural Science Foundation(ZR202210270038)。

摘　　要：Alfalfa(Medicago sativa.L.)is a globally significant autotetraploid legume forage crop.However,despite its importance,establishing efficient gene editing systems for cultivated alfalfa remains a formidable challenge.In this study,we pioneered the development of a highly effective ultrasonic-assisted leaf disc transformation system for Gongnong 1 alfalfa,a variety widely cultivated in Northeast China.Subsequently,we created a single transcript CRISPR/Cas9(CRISPR_2.0)toolkit,incorporating multiplex gRNAs,designed for gene editing in Gongnong 1.Both Cas9 and gRNA scaffolds were under the control of the Arabidopsis ubiquitin-10 promoter,a widely employed polymeraseⅡconstitutive promoter known for strong transgene expression in dicots.To assess the toolkit’s efficiency,we targeted PALM1,a gene associated with a recognizable multifoliate phenotype.Utilizing the CRISPR_2.0 toolkit,we directed PALM1 editing at two sites in the wild-type Gongnong 1.Results indicated a 35.1%occurrence of editing events all in target 2 alleles,while no mutations were detected at target 1 in the transgenic-positive lines.To explore more efficient sgRNAs,we developed a rapid,reliable screening system based on Agrobacterium rhizogenes-mediated hairy root transformation,incorporating the visible reporter MtLAP1.This screening system demonstrated that most purple visible hairy roots underwent gene editing.Notably,sgRNA3,with an 83.0%editing efficiency,was selected using the visible hairy root system.As anticipated,tetra-allelic homozygous palm1 mutations exhibited a clear multifoliate phenotype.These palm1 lines demonstrated an average crude protein yield increase of 21.5%compared to trifoliolate alfalfa.Our findings highlight the modified CRISPR_2.0 system as a highly efficient and robust gene editing tool for autotetraploid alfalfa.

关 键 词：ALFALFA Gene editing CRISPR_2.0 toolkit Hairy root system Tetra-allelic homozygous mutants 

分 类 号：S541.9[农业科学—作物学] Q943.2[生物学—植物学]