A complete CRISPR-Cas9-mediated knockout of the BEL5 gene, encoding a transcription factor, is reported, known as one of the key regulators driving tuber formation, and a regulatory role of BEL5 in the timing of tuber onset but, unexpectedly, its dispensability for tuber development in modern cultivated potato is proposed.
Abstract
CRISPR-Cas9 has emerged as a powerful tool for targeted genome editing in plants; however, its application in tetraploid potato (Solanum tuberosum ssp. tuberosum) remains challenging due to its vegetative propagation and complex highly heterozygous genome. Availability of whole-genome sequence data for the specific genotype is crucial to ensure complete knockout of all alleles of target genes while minimizing off-target mutations. In this study, using the tetraploid potato cultivar Désirée, we report, a complete CRISPR-Cas9-mediated knockout of the BEL5 gene, encoding a transcription factor, known as one of the key regulators driving tuber formation. We employed Agrobacterium-mediated transformation and demonstrated that repeated de novo regeneration could improve editing efficiency by promoting emergence of new mutations. BEL5 knockout plants exhibited a delayed onset of tuberization under inductive short-day conditions in hydroponics; however, their overall tuber yields were comparable to wild type plants. Based on our results, we propose a regulatory role of BEL5 in the timing of tuber onset but, unexpectedly, its dispensability for tuber development in modern cultivated potato. Besides providing functional insight into the BEL5 role in potato, this study includes a methodological approach for efficient CRISPR-Cas9 gene editing in this vegetatively propagated polyploid crop, along with strategies for detecting mutations in genes that lack clear phenotypic manifestation.
iPB-REG is established as a practical strategy for producing uniform genome-edited fruit trees and provide a valuable platform for DNA-free genetic improvement and functional genomics in clonally propagated perennial crops.
C. Nishitani, Nozomi Tsujino, Misa Kuroki et al.· bioRxiv· 0 citations
Sugarbeet (Beta vulgaris ssp. vulgaris, L.), is a vital temperate crop, supplying nearly 40% of the world’s sugar. However, its high susceptibility to bacterial, fungal, and viral diseases creates an urgent need for improved, disease-resistant cultivars. The CRISPR/Cas9 system has rapidly advanced plant genetic engineering by enabling precise and targeted genome modifications. Our goal is to establish a gene-editing platform in sugarbeet to support future development of disease-resistant lines by targeting the candidate genes. In this study, we applied CRISPR/Cas9 to generate targeted mutations in two genes involved in chlorophyll biosynthesis and carotenoid-mediated leaf pigmentation: magnesium chelatase (Mg-chelatase) and phytoene desaturase (PDS). Two CRISPR/Cas9 constructs, each carrying an sgRNA targeting either Mg-chelatase or PDS, were developed and mobilized into Agrobacterium tumefaciens. A total of 233 and 200 hypocotyl explants were transformed with constructs targeting Mg-chelatase and PDS, resulting in regeneration efficiencies of 8% and 14% on kanamycin selection medium, respectively. Light green, yellow, variegated yellow-green, and albino phenotypes were observed among the putative transformants, whereas non-edited transformed lines resembled untransformed control plants. Targeted mutations, including insertions, deletions, and substitutions of nucleotides, were identified at both genomic loci, with editing efficiencies of 60.0% for Mg-chelatase and 68.75% for PDS underscoring the effectiveness of this approach in sugarbeet, a recalcitrant crop. Deletions ranged from 5 to 28 bp in Mg-chelatase and 2 to 21 bp in PDS, while insertion events consisted of single-base additions in Mg-chelatase edited lines and larger insertions of 7–16 bp in PDS mutants. The results demonstrate the successful deployment of CRISPR/Cas9 for targeted genome engineering in sugarbeet and establish a reliable platform for future gene-editing efforts aimed at enhancing resistance to a wide range of pathogens and diseases affecting the crop.
Z. Khan, Tinley Hathaway, C. Chu et al.· Frontiers in Genome Editing· 0 citations
Rice (Oryza sativa L.) is a staple food crop worldwide, and improving disease resistance is a core target in rice breeding. In this study, we employed CRISPR/Cas9 genome editing to modify the coding sequence (CDS) of two susceptibility genes, Bsr-d1 and Pi21, in the elite maintainer line Gengxiang B to enhance its blast resistance. We generated Bsr-d1/Pi21 double homozygous mutants via Agrobacterium-mediated genetic transformation. Quantitative RT-PCR revealed significantly suppressed transcript accumulation of both target genes in the edited lines compared with the wild type Gengxiang B. Upon inoculation with Magnaporthe oryzae, multiple defense-related marker genes were markedly upregulated in the double mutants. Phenotypic assays demonstrated significantly reduced disease severity for both leaf and panicle blast in the edited lines compared with the wild type. Importantly, no statistically detectable differences were found between the double mutants and wild-type plants for key agronomic or grain quality traits. Collectively, these results demonstrate that CRISPR/Cas9-mediated editing of susceptibility loci generates genetically stable blast-resistant rice germplasm without compromising agronomic traits or grain quality, providing valuable genetic resources for future rice varietal improvement.
Ke Lan, Lin Yuan, Da-Cheng Zhao et al.· Plants· 0 citations