The development of the Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas9) system has advanced genome editing and has become widely adopted for this purpose in many species. Its efficient use requires the method adjustment and optimization. Here, we show the use of a model carrot callus system for demonstrating gene editing via CRISPR/Cas9 targeted mutagenesis. The system relies on the utilization of carrot tissue accumulating anthocyanin pigments responsible for a deep purple cell color and generation of knockout mutations in the flavanone-3-hydroxylase (F3H) gene in the anthocyanin biosynthesis pathway. F3H mutant cells targeted by Cas9/gRNA complexes are not able to synthesize anthocyanins and remain white, easily visually distinguished from purple wild-type cells. Mutations are either small indels or larger chromosomal deletions that can be identified by restriction fragment analysis and sequencing. This feasible system can also be applied for validating efficiency of CRISPR/Cas9 vectors.
Keywords: Agrobacterium-mediated transformation; Anthocyanins; Callus; Daucus carota; F3H; Flavanone-3-hydroxylase.