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Efficient gene editing of a model fern species through gametophyte-based transformation

文献类型： 外文期刊

作者： Jiang, Wei 1 ; Deng, Fenglin 1 ; Babla, Mohammad 2 ; Chen, Chen 2 ; Yang, Dongmei 2 ; Tong, Tao 1 ; Qin, Yuan 1 ; Chen, Guang 6 ; Marchant, Blaine 2 ; Soltis, Pamela 8 ; Soltis, Douglas Edward 8 ; Zeng, Fanrong 1 ; Chen, Zhong-Hua 2 ;

作者机构： 1.Yangtze Univ, Coll Agr, MARA Key Lab Sustainable Crop Prod Middle Reaches, Jingzhou 434025, Peoples R China

2.Western Sydney Univ, Sch Sci, Penrith, NSW 2751, Australia

3.Xianghu Lab, Hangzhou 311231, Peoples R China

4.Hubei Hongshan Lab, Wuhan 430070, Peoples R China

5.Hainan Univ, Sch Trop Agr & Forestry, Danzhou 571737, Peoples R China

6.Zhejiang Acad Agr Sci, Inst Digital Agr, Hangzhou 310021, Peoples R China

7.Univ Missouri, Dept Biol, St Louis, MO 63121 USA

8.Univ Florida, Dept Biol, Gainesville, FL 32611 USA

9.Western Sydney Univ, Hawkesbury Inst Environm, Penrith, NSW 2751, Australia

期刊名称：PLANT PHYSIOLOGY （ 影响因子：6.9； 五年影响因子：7.7 ）

ISSN： 0032-0889

年卷期： 2024 年 196 卷 4 期

页码：

收录情况： SCI

摘要： The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 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 through gametophytes due to the complex characteristics of fern genomes, genetics, and physiology. Here, we established a protocol for gametophyte-based screening of single-guide RNAs (sgRNAs) with high efficiency for CRISPR/Cas9-mediated gene knockout in a model fern species, Ceratopteris richardii. We utilized the C. richardii ACTIN promoter to drive sgRNA expression and the 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, such as Nucleotidase/phosphatase 1 (CrSAL1) and Phytoene Desaturase (CrPDS), which resulted in an albino phenotype in C. richardii. Knockout of CrSAL1 resulted in significantly (P < 0.05) reduced stomatal conductance (g(s)), leaf transpiration rate (E), guard cell 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), g(s), and E as well as most of the stomatal traits and ABA-induced ROS production in guard cells compared to the wild-type (WT) plants. Taken together, our 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.

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