Efficient Gene Editing and Overexpression of Gametophyte Transformation in a Model Fern
Efficient Gene Editing and Overexpression of Gametophyte Transformation in a Model Fern
Abstract The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-related nuclease (Cas) system allows precise and easy editing of genes in many plant species. However, this system has not yet been applied to any fern species due to the complex characteristics of fern genomes, genetics and physiology. Here, we established, for the first time, a protocol for gametophyte-based screening single-guide RNAs (sgRNAs) with high efficiency for CRISPR/Cas-mediated gene editing in a model fern species, Ceratopteris richardii. We utilized the C. richardii Actin promoter to drive sgRNA expression and enhanced CaMV 35S promoter to drive the expression of Streptococcus pyogenes Cas9 in this CRISPR-mediated editing system, which was employed to successfully edit a few genes (e.g., nucleotidase/phosphatase 1, CrSAL1; Cryptochrome 4, CRY4) and CrPDS, encoding a phytoene desaturase protein that resulted in an albino phenotype in C. richardii. Knockout of CrSAL1 resulted in significantly reduced stomatal conductance (gs), leaf transpiration rate (E), stomatal/pore length, and abscisic acid (ABA)-induced reactive oxygen species (ROS) accumulation in guard cells. Moreover, CrSAL1 overexpressing plants showed significantly increased net photosynthetic rate (A), gs, E and intrinsic water use efficiency (iWUE) as well as most of the stomatal traits and ROS production in guard cells compared to those in the wild-type (WT) plants. Taken together, the optimized CRISPR/Cas9 system provides a useful tool for functional genomics in a model fern species, allowing the exploration of fern gene functions for evolutionary biology, herbal medicine discovery and agricultural applications.
Jiang Wei、Tong Tao、Qin Yuan、Chen Zhong-Hua、Marchant D. Blaine、Soltis Pamela S.、Chen Chen、Yang Dongmei、Soltis Douglas E.、Deng Fenglin、Babla Mohammad、Chen Guang、Zeng Fanrong
MARA Key Laboratory of Sustainable Crop Production in the Middle Reaches of the Yangtze River, College of Agriculture, Yangtze University||School of Science, Western Sydney UniversityMARA Key Laboratory of Sustainable Crop Production in the Middle Reaches of the Yangtze River, College of Agriculture, Yangtze UniversityMARA Key Laboratory of Sustainable Crop Production in the Middle Reaches of the Yangtze River, College of Agriculture, Yangtze UniversitySchool of Science, Western Sydney University||Hawkesbury Institute for the Environment, Western Sydney UniversityBiology Department, University of Missouri at St. LouisDepartment of Biology, University of FloridaSchool of Science, Western Sydney UniversitySchool of Science, Western Sydney UniversityDepartment of Biology, University of FloridaMARA Key Laboratory of Sustainable Crop Production in the Middle Reaches of the Yangtze River, College of Agriculture, Yangtze UniversitySchool of Science, Western Sydney UniversityCentral Laboratory, Zhejiang Academy of Agricultural ScienceMARA Key Laboratory of Sustainable Crop Production in the Middle Reaches of the Yangtze River, College of Agriculture, Yangtze University
遗传学分子生物学植物学
CRISPR/Casgenetic transformationoverexpressiongene functionCeratopteris richardii
Jiang Wei,Tong Tao,Qin Yuan,Chen Zhong-Hua,Marchant D. Blaine,Soltis Pamela S.,Chen Chen,Yang Dongmei,Soltis Douglas E.,Deng Fenglin,Babla Mohammad,Chen Guang,Zeng Fanrong.Efficient Gene Editing and Overexpression of Gametophyte Transformation in a Model Fern[EB/OL].(2025-03-28)[2025-05-22].https://www.biorxiv.org/content/10.1101/2024.04.10.588889.点此复制
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