Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice

TLDR

The type II CRISPR/Cas system from *Streptococcus pyogenes* and its simplified Cas9/sgRNA derivative have emerged as powerful tools for targeted gene knockout in diverse organisms, including bacteria, yeast, fruit fly, zebrafish, and human cells. Here, we adapted these systems to achieve successful expression of Cas9/sgRNA in the dicot plants *Arabidopsis* and tobacco and the monocot crops rice and sorghum. Agrobacterium tumefaciens delivered Cas9, sgRNA, and a mutant GFP gene containing Cas9 target sites into *Arabidopsis* and tobacco, where Cas9/sgRNA cleavage and error‑prone repair restored functional GFP expression. Sequencing confirmed Cas9/sgRNA‑mediated mutations at target sites in Arabidopsis, tobacco, and rice protoplasts, demonstrating the system’s effectiveness for plant genetic engineering.

Abstract

The type II CRISPR/Cas system from Streptococcus pyogenes and its simplified derivative, the Cas9/single guide RNA (sgRNA) system, have emerged as potent new tools for targeted gene knockout in bacteria, yeast, fruit fly, zebrafish and human cells. Here, we describe adaptations of these systems leading to successful expression of the Cas9/sgRNA system in two dicot plant species, Arabidopsis and tobacco, and two monocot crop species, rice and sorghum. Agrobacterium tumefaciens was used for delivery of genes encoding Cas9, sgRNA and a non-fuctional, mutant green fluorescence protein (GFP) to Arabidopsis and tobacco. The mutant GFP gene contained target sites in its 5′ coding regions that were successfully cleaved by a CAS9/sgRNA complex that, along with error-prone DNA repair, resulted in creation of functional GFP genes. DNA sequencing confirmed Cas9/sgRNA-mediated mutagenesis at the target site. Rice protoplast cells transformed with Cas9/sgRNA constructs targeting the promoter region of the bacterial blight susceptibility genes, OsSWEET14 and OsSWEET11 , were confirmed by DNA sequencing to contain mutated DNA sequences at the target sites. Successful demonstration of the Cas9/sgRNA system in model plant and crop species bodes well for its near-term use as a facile and powerful means of plant genetic engineering for scientific and agricultural applications.