Target-Directed Mutation Using a RGEN Ribonucleoproteins (RNPs) and sgRNA in a Model Plant Petunia

Recently cloning free CRISPR/Cas system has been applied to plant cells and experiments using direct delivery of RGEN ribonucleoproteins (RNPs and target specific in vitro transcribed sgRNA. Petunia (Petunia hybrida) protoplasts were isolated from 20-day-old in vitro grown seedlings after germination in MS medium. gRNAs were carefully prepared for 6 target sites which have higher out-of-frame scores for higher complete knock out efficiency in the coding regions of nitrate reductatse (NR) gene locus. The higher yield of protoplasts was obtained with cellulose and macerozyme enzymes, up to 1.6 × 10⁶ protoplasts for 4–7 hours of incubation. After transformation, fluorescence microscopic analyses revealed that approximately 50% and 90% of transformed protoplasts had GFP expression after 24 and 72 h of incubation, respectively. The mutation frequencies of Indel ranged from 2.4 to 20% in successfully amplified four NR targets, which was also confirmed using T7E1 assay. The average mutation rate obtained at five different sites in NR gene locus was 11.5 ± 2%. Our study demonstrated that direct delivery of engineered RGEN RNPs is an effective breeding tool for Petunia and possibly many other crops in a target-specific manner.