CRISPR/Cas9-mediated multiple gene editing in Brassica oleracea var. capitata using the endogenous tRNA-processing system

Abstract

Cabbage (Brassica oleracea var. capitata) is a biennial plant with strong self-incompatibility and an obligate requirement for prolonged vernalization by exposure to low temperatures to induce flowering. These characteristics significantly increase the difficulty of exploiting novel germplasm induced by physical or chemical mutagens. In this study, we report a CRISPR/Cas9 gene-editing system based on endogenous tRNA processing to induce high efficiency and inheritable mutagenesis in cabbage. Using the phytoene desaturase gene BoPDS, the S-receptor kinase gene BoSRK, and the male-sterility-associated gene BoMS1 as the target genes, multisite and multiple gene mutations were achieved using a construct with tandemly arrayed tRNA-sgRNA architecture to express multiple sgRNAs. The BoSRK3 gene mutation suppressed self-incompatibility completely, converting the self-incompatible line into a self-compatible line. In addition, the BoMS1 gene mutation produced a completely male-sterile mutant, which was highly cross compatible with its nonmutant isoline at the flowering stage as a result of a simultaneous BoSRK3 gene mutation, enabling the economic propagation of the male-sterile line through bee-mediated cross-pollination. Interestingly, higher site mutation efficiency was detected when a guide sequence was inserted into a location in the tandemly arrayed tRNA-sgRNA architecture that was distal from the upstream Pol III promoter. In addition, mutation sites were also detected in the paralogous genes of the BoPDS and BoSRK genes that had fully consistent sequences or base mismatches but beyond the “seed” region in the spacer sequence compared with the target sgRNAs. Collectively, our results demonstrate that the CRISPR/Cas9 system, coupled with an endogenous tRNA-processing system, is an efficient tool to improve cabbage traits.

Elucidating the roles of three β-glucuronosyltransferases (GLCATs) acting on arabinogalactan-proteins using a CRISPR-Cas9 multiplexing approach in Arabidopsis

“…Traditional genetic crossing/breeding approaches to obtain higher order mutants, however, are both time-consuming and complicated by genetic linkage. Compared to traditional breeding, CRISPR-Cas9 gene editing has been recently used to study redundant gene families in several plant species such as rice, wheat, cabbage, tomato and Arabidopsis [21,[26][27][28][29]. The research reported here, however, represents the first application of CRISPR/Cas9 to understand GTs families in Arabidopsis.…”

Advances in molecular biology of Paeonia L.

“…CRISPR/Cas9 enables precise modification of genomic sequences and the ability to regulate gene expression patterns (Mahfouz et al 2014). The application of CRISPR/ Cas9 has been increasing in plants (Liu et al 2019b;Ma et al 2019;Zhang et al 2019b). Liu et al (2019b) used the CRISPR/Cas9 genome-editing system based on RNA endoribonuclease Csy4 processing to induce high-efficiency and inheritable targeted deletion of genes (AP1, SVP, and TFL1) involved in floral development in A. thaliana.…”

“…Zhang et al (2019b) used the CRISPR/Cas9 system to achieve efficient mutagenesis of five wheat genes, confirming that CRISPR/Cas9-induced gene editing plays important roles in wheat genome engineering through Agrobacteriummediated transformation. Based on the CRISPR/Cas9 gene editing system, Ma et al (2019) achieved trait stacking in cabbage (Brassica oleracea) with the phytoene desaturase gene BoPDS, the S-receptor kinase gene BoSRK and the male-sterility-related gene BoMS1 as target genes. This confirmed that CRISPR/CAS9 is an efficient tool for studying gene function.…”

An update on the arsenal: mining resistance genes for disease management of Brassica crops in the genomic era

“…Notably, the quickly emerging gene-editing technique helps realize accurate alteration of the target DNA sequence. Ma et al 172 applied CRISPR/Cas9-mediated multiple gene editing in cabbage, with the targets BoPDS, BoSRK, and BoMS1, and successfully generated albino, self-compatible, and male sterile lines, showing its great power in improving plant traits.…”

“…f Pyramiding both the qualitative and quantitative R loci generates durable BL resistance in B. napus74 . g CRISPR/Cas9based gene editing helps knockout multiple target genes in B. oleracea172 . h Expressing the CP gene from TuMV confers high resistance in B. napus200…”