Multiplexed CRISPR/Cas9-mediated metabolic engineering of γ-aminobutyric acid levels in Solanum lycopersicum

In recent years, the type II CRISPR system has become a widely used and robust technique to implement site-directed mutagenesis in a variety of species including model and crop plants. However, few studies manipulated metabolic pathways in plants using the CRISPR system. Here, we introduced the pYLCRISPR/Cas9 system with one or two single-site guide RNAs to target the tomato phytoene desaturase gene. An obvious albino phenotype was observed in T0 regenerated plants, and more than 61% of the desired target sites were edited. Furthermore, we manipulated the gamma-aminobutyric acid (GABA) shunt in tomatoes using a multiplex pYLCRISPR/Cas9 system that targeted five key genes. Fifty-three genome-edited plants were obtained following single plant transformation, and these samples represented single to quadruple mutants. The GABA accumulation in both the leaves and fruits of genomically edited lines was significantly enhanced, and the GABA content in the leaves of quadruple mutants was 19-fold higher than that in wild-type plants. Our data demonstrate that the multiplex CRISPR/Cas9 system can be exploited to precisely edit tomato genomic sequences and effectively create multisite knockout mutations, which could shed new light on plant metabolic engineering regulations.