Site-specific editing of the cysteine transferase (CYS) gene in tobacco using the CRISPR/Cas9 technology

Abstract

In order to study the effects of cysteine transferase (CYS) gene on the growth, development and anthocyanin synthesis in tobacco, we established CRISPR/Cas9 gene editing system in tobacco and constructed a double target CRISPR/Cas9 gene editing vector P35S-H-CYS. CYS tobacco mutant lines were obtained by Agrobacterium- mediated genetic system, the CYS gene knockout effect was tested by base sequencing and RTqPCR, the mutation types of target genes were analyzed. Sequencing results showed that 3 mutant lines had gene mutations at target sites, including base insertion and base replacement. Target site 1 could bind to target site more effectively, and target site 2 produced two bases mutations, both synonymous mutations of amino acids. RTqPCR results showed that the relative expression level of CYS gene in mutants were significantly lower than the wild type tobacco (P< 0.01). Compared to wild type, the multiple of gene transcription levels were 0.31, 0.45 and 0.34. The knockout ofCYS gene had no significant effect on tobacco plant height and leaf shape, but the anthocyanin content in the mutant tobacco were increased in different degrees. The content of anthocyanin in CYS-1 mutant was 1.3-fold, which was significantly higher than that of wild type (P<0.05). These results indicated that CYS gene can inhibit anthocyanin synthesis, which was consistent with the previous transcriptome analysis results. In this study, CRISPR/ Cas9 gene editing technology was used to design two target sites for targeted editing of CYS gene. Using tobacco as the transformation receptor provided an important material basis for further study of the function of CYS.