Efficient Gene Editing and Overexpression of Gametophyte Transformation in a Model Fern

Abstract

AbstractThe 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 theC. richardii Actinpromoter to drive sgRNA expression and enhanced CaMV 35S promoter to drive the expression ofStreptococcus pyogenesCas9 in this CRISPR-mediated editing system, which was employed to successfully edit a few genes (e.g.,nucleotidase/phosphatase 1, CrSAL1;Cryptochrome 4, CRY4) andCrPDS, encoding a phytoene desaturase protein that resulted in an albino phenotype inC. richardii. Knockout ofCrSAL1resulted 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,CrSAL1overexpressing plants showed significantly increased net photosynthetic rate (A),gs, Eand 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.