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

Affiliations
  • 1MARA Key Laboratory of Sustainable Crop Production in the Middle Reaches of the Yangtze River, College of Agriculture, Yangtze University, Jingzhou 434025, China.
  • 2School of Science, Western Sydney University, Penrith, NSW 2751, Australia.
  • 3Xianghu Laboratory, Hangzhou 311231, China.
  • 4Hubei Hongshan Laboratory, Wuhan, 430070, China.
  • 5School of Tropical Agriculture and Forestry, Hainan University, Danzhou, 571737, China.
  • 6Institute of Digital Agriculture, Zhejiang Academy of Agricultural Science, Hangzhou 310021, China.
  • 7Department of Biology, University of Missouri-St. Louis, St. Louis, MO 63121, USA.
  • 8Department of Biology, University of Florida, Gainesville, FL 32611, USA.
  • 9Hawkesbury Institute for the Environment, Western Sydney University, Penrith, NSW 2751, Australia.

Published on:

September 13, 2024

Abstract

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 (gs), 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), gs, 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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