CRISPR/Cas9-mediated mutagenesis of homologous genes in Chinese kale

Abstract

The clustered regulatory interspaced short palindromic repeat-associated protein 9 (CRISPR/Cas9) system has developed into a powerful gene-editing tool that has been successfully applied to various plant species. However, studies on the application of the CRISPR/Cas9 system to cultivated Brassica vegetables are limited. Here, we reported CRISPR/Cas9-mediated genome editing in Chinese kale (Brassica oleracea var. alboglabra) for the first time. A stretch of homologous genes, namely BaPDS1 and BaPDS2, was selected as the target site. Several stable transgenic lines with different types of mutations were generated via Agrobacterium-mediated transformation, including BaPDS1 and BaPDS2 double mutations and BaPDS1 or BaPDS2 single mutations. The overall mutation rate reached 76.47%, and these mutations involved nucleotide changes of fewer than 10 bp. The clear albino phenotype was observed in all of the mutants, including one that harbored a mutation within an intron region, thereby indicating the importance of the intron. Cleavage in Chinese kale using CRISPR/Cas9 was biased towards AT-rich sequences. Furthermore, no off-target events were observed. Functional differences between BaPDS1 and BaPDS2 were also assessed in terms of the phenotypes of the respective mutants. In combination, these findings showed that CRISPR/Cas9-mediated targeted mutagenesis can simultaneously and efficiently modify homologous gene copies of Chinese kale and provide a convenient approach for studying gene function and improving the yield and quality of cultivated Brassica vegetables.

Color-related chlorophyll and carotenoid concentrations of Chinese kale can be altered through CRISPR/Cas9 targeted editing of the carotenoid isomerase gene BoaCRTISO

“…The transformation of Chinese kale was performed as described in our previous study 19 . Explants were precultured for 3 days in MS media consisting of 0.5 mg L −1 2,4-D and 0.8% Phytagar, after which the explants were infected with the Agrobacterium strain GV3101 by immersion for 1-2 min.…”

“…Approximately 10 monoclones of each mutant were detected by sequencing. The PCR procedure was as follows: denaturation at 95°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 30 s. The transformants with the hygromycin gene were used for further analysis 19 .…”

“…The PCR products were then sequenced for genotypic analysis of the transgenic plants. The mutation rate was calculated, and all sequencing data were collected to analyze the mutation type 19 . Amino acid changes in the mutants were analyzed by DNAMAN 6.0 (Lynnon Biosoft, California, USA).…”

“…Recently, we established a CRISPR/Cas9 gene editing system in Chinese kale 19 and used it to demonstrate functional differences among members of the PDS family, which are important genes involved in the carotenoid biosynthetic pathway 20 . Chinese kale is not typically colorful: the edible organs of most varieties are green, except for a few varieties that have red bolting stems 21 .…”

Establishment of Efficient Genetic Transformation Systems and Application of CRISPR/Cas9 Genome Editing Technology in Lilium pumilum DC. Fisch. and Lilium longiflorum White Heaven

“…To quickly verify the effects of CRISPR/Cas9 in plants, the PDS gene encoding phytoene desaturase, a key enzyme of carotenoid synthesis, is often targeted as a reporter gene due to the obvious albinism and dwarfism caused by its loss of function as evidenced in Arabidopsis [37], Citrus sinensis [38], Populus [39], Petunia hybrida [40], Nicotiana benthamiana [41], Medicago truncatula [42], Manihot esculenta Crantz [43], Triticum aestivum L. [44] Actinidia Lindl. [33], Brassica oleracea [45], Malus x domestica Bork. and Pyrus communis L. [46].…”