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.
Highlights
Methods for rapidly and efficiently editing plant genomes are useful for research into gene function and crop improvement
Our results demonstrate that the clustered regularly interspaced short palindromic repeats (CRISPR)/ Cas[9] system with endogenous tRNA processing is a powerful tool to improve cabbage varieties using specific gene modification
The BoPDS gene, the self-incompatibility gene BoSRK3, the BoMS1 gene associated with male sterility, and some of their paralogous genes were successfully knocked out using an array of single-guide RNAs (sgRNAs)-tRNA units designed to express more than one sgRNA and showed obvious mutant phenotypes in the T0 generation
Summary
Methods for rapidly and efficiently editing plant genomes are useful for research into gene function and crop improvement. The most common strategy is to stack multiple independent sgRNA-expressing cassettes in a single construct and to deliver the construct into plant cells[6,8]. This sgRNA-expressing strategy is challenging for most organisms due to the limitations of the delivery method and the vector capacity. By using a synthetic gene with tandemly arrayed tRNA-sgRNA architecture, multiple sgRNAs were efficiently and precisely produced in vivo in rice by the endogenous RNases, RNase P and Z, for the first time[9]. The tRNA-processing sgRNA expression system was successfully demonstrated in other organisms[10,11,12,13]
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