Development of a single transcript CRISPR/Cas9 toolkit for efficient genome editing in autotetraploid alfalfa
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
Affiliations
Haixia Zhao
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; Shandong Energy Institute, Qingdao 266101, Shandong, China; Qingdao New Energy Shandong Laboratory, Qingdao 266101, Shandong, China; University of Chinese Academy of Sciences, Beijing 100049, China
Siyi Zhao
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; The Key Laboratory of Plant Development and Environmental Adaptation Biology, Ministry of Education, School of Life Sciences, Shandong University, Qingdao 266101, Shandong, China
Yingping Cao
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; Shandong Energy Institute, Qingdao 266101, Shandong, China; Qingdao New Energy Shandong Laboratory, Qingdao 266101, Shandong, China; University of Chinese Academy of Sciences, Beijing 100049, China
Xiping Jiang
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
Lijuan Zhao
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; School of Ecology Environment, Ningxia University, Yinchuan 750021, Ningxia, China
Zhimeng Li
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
Mengqi Wang
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
Ruijuan Yang
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; Shandong Energy Institute, Qingdao 266101, Shandong, China; Qingdao New Energy Shandong Laboratory, Qingdao 266101, Shandong, China
Chuanen Zhou
The Key Laboratory of Plant Development and Environmental Adaptation Biology, Ministry of Education, School of Life Sciences, Shandong University, Qingdao 266101, Shandong, China
Zhaoming Wang
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
Feng Yuan
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
Dongmei Ma
School of Ecology Environment, Ningxia University, Yinchuan 750021, Ningxia, China
Hao Lin
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China
Wenwen Liu
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; Shandong Energy Institute, Qingdao 266101, Shandong, China; Qingdao New Energy Shandong Laboratory, Qingdao 266101, Shandong, China; Corresponding authors.
Chunxiang Fu
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; Shandong Energy Institute, Qingdao 266101, Shandong, China; Qingdao New Energy Shandong Laboratory, Qingdao 266101, Shandong, China; University of Chinese Academy of Sciences, Beijing 100049, China; Corresponding authors.
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 polymerase II constitutive 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.
