CRISPR-Cas9 Targeted Mutagenesis of Green Fluorescent Protein Transgene and a Native, Granule Bound Starch Synthase, Gene in Potato

Abstract

Potato (Solanum tuberosum L.) was genetically modified using the CRISPR-Cas9 system, first to evaluate its efficacy in knocking out a genome-integrated transgene and then to alter its starch profile. The CRISPR system, which acts as an adaptive immune system in Streptococcus pyogenes, is being used to engineer eukaryotic genomes in a precise manner. It has proven to be a simple, yet efficient, tool to generate targeted mutations. Potato is an important crop that serves as a staple food for 1.3 billion people and is also a source of starch for industrial, non-food purposes. We first used transgenic potato plants that contained either a single-copy green fluorescence protein (gfp) gene insert or four different gfp gene inserts to evaluate the efficacy of the CRISPR-Cas9 system to disrupt a target gene integrated in the genome of this crop. Three sgRNAs were examined for targeted knock out of this gene. In combination with Cas9, all three sgRNAs successfully knocked out the gene, however their efficiencies differed greatly. The nature of mutations was analyzed from various knockout events. Once the technology and protocols were established, the second phase of the project involved disruption of the granule-bound starch synthase (gbssI) gene using two different sgRNAs. The objective was to modify starch profile by eliminating amylose in the tuber. Lugol-iodine staining of the tuber showed amylose reduction in various edited events and these results were confirmed using both the perchloric acid and enzymatic method. One event out of six examined showed a complete knock out of all gbss alleles and total elimination of amylose from the tuber. Viscosity profiles of the tuber starch from different events were determined using Rapid Visco Analyzer (RVA). The results further confirmed the alterations in starch composition in the tubers from these events.