Carrot genome editing using CRISPR-based systems

Abstract

Genome editing using CRISPR-based tools is a breakthrough technology allowing the generation of mutations precisely in the indicated target DNA sequence. In this review we present the principles of genome editing using the CRISPR/Cas9 system and its application to research in carrot, one of the most important horticultural crops. We summarize experimental results in validation of the system by knocking out the flavanone 3-hydroxylase (F3H) gene critical for anthocyanins biosynthesis. Using a model callus tissue accumulating high level of these pigments it was shown that CRISPR/Cas9 vectors can be designed to target the F3H gene and generate mutations leading to non-functional enzyme hence blockage of the anthocyanin biosynthesis and tissue discoloration. The system was then used to knock out various genes related to carotenoid biosynthesis like the gene coding for geranylgeranyl diphosphate reductase (GGred) which redirects carotenoid precursors to phytyl chain pathway, or lycopene e-cyclase (LCYE), a key enzyme of α-carotene biosynthesis. It was also used to generate haploid inducers by knocking out the centromeric histone H3 (CENH3) gene. Various types of mutations generated in the carrot genome using CRISPR/Cas9 vectors confirmed that it is a feasible method to induce changes precisely in the desired target genes.