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

文献类型： 外文期刊

第一作者： Zhao, Haixia

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

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关键词： Alfalfa; Gene editing; CRISPR_2.0 toolkit; Hairy root system; Tetra -allelic homozygous mutants

期刊名称：CROP JOURNAL （ 影响因子：6.0； 五年影响因子：5.6 ）

ISSN： 2095-5421

年卷期： 2024 年 12 卷 3 期

页码：

收录情况： SCI

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 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. (c) 2024 Crop Science Society of China and Institute of Crop Science, CAAS. Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NCND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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