Efficient genome editing of rubber tree (hevea brasiliensis) protoplasts using CRISPR/Cas9 ribonucleoproteins

Abstract CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) is a powerful targeted mutagenesis tool that has been implemented in many plant species. To date, the application of CRISPR/Cas9 in rubber tree (Hevea brasiliesis) has not yet been reported. Here, we describe the efficient targeted mutagenesis in rubber tree by direct delivery of CRISPR/Cas9 ribonucleoproteins (RNPs). Five sgRNAs were designed to target FLOWERING LOCUS T (FT) and TERMINAL FLOWER1 (TFL1) genes in rubber tree. Using a Cas9/sgRNA ratio of 1:7, the Cas9 proteins were preassembled with in vitro transcribed sgRNAs, and then introduced into rubber tree protoplasts. Targeted mutations were successfully induced at frequencies ranging from 3.74% to 20.11% at five target sites. Two mutation patterns including +1 nt insertions and deletions were detected at all target sites, and the -1 nt deletion was the most common mutation obtained in all cases. In addition, by delivering combinations of sgRNAs targeting multiple genes, multiple targeted mutations were induced in rubber tree protoplasts through one transformation step. This RNP-based genome editing system demonstrates the potential for precise genetic modifications of rubber tree. Furthermore, together with the rubber tree protoplast regeneration system, our study provides a promising approach for the production of DNA-free genome edited rubber tree plants from protoplast.