ویرایش ژن هیستون سانترومری CenH3 در گیاه خیار با استفاده از فن‌آوری کریسپر

Introduction
Cucumber (Cucumis sativus L.) is one of the most economically important crops among the Cucurbitaceae family. The development of high-quality hybrid cucumber varieties has greatly contributed to early maturity, enhanced drought resistance, better fruit uniformity, and increased yield. These hybrids are typically produced using homozygous pure lines, which are traditionally generated through recurrent selfing over multiple generations—a time-consuming and labor-intensive process. However, recent advances in genome editing have introduced a transformative pathway: the induction of haploids and doubled haploids , offering a rapid route to completely homozygous lines within one or two generations. While haploid and doubled haploid production has been reported in various crop species, the Cucurbitaceae family has lagged behind, with only limited successful applications of CRISPR/Cas9 technology for haploid induction. In particular, the application of CRISPR to target the centromeric histone H3 gene (CenH3), a gene with a pivotal role in chromosome segregation during cell division, remains largely unexplored in cucumber. This study aimed to investigate the feasibility of using CRISPR/Cas9 to induce targeted mutations in the CenH3 gene in cucumber. The long-term objective was to assess whether such mutations could serve as a basis for developing haploid inducer lines—a vital tool in modern cucumber breeding programs.
 
Materials and Methods
To design a targeted genome editing construct, CenH3 sequences from cucumber and Arabidopsis thaliana were aligned using the Histone Database (HPD). A conserved motif (ALQEAAE) within the histone fold domain was identified, and a specific guide RNA (gRNA) targeting the alanine codon within this region was selected. The corresponding single-guide RNA (sgRNA) was cloned into the pDE-Cas9 vector, which was subsequently introduced into Agrobacterium rhizogenes to facilitate plant transformation. A composite plant system was employed, whereby cucumber seedlings were inoculated with A. rhizogenes carrying the CRISPR construct. Hairy root formation was induced, and genomic DNA was extracted from transformed root tissues. PCR amplification and DNA sequencing were used to confirm the presence of the CRISPR construct and to detect mutations in the target site of the CenH3 gene.
 
Results
From a total of 700 inoculated cucumber explants, approximately 70% developed hairy roots. PCR screening confirmed successful integration of the CRISPR/Cas9 construct in 10 samples. DNA sequencing of three positive samples revealed three distinct mutations in the CenH3 gene, all within 11 nucleotides of the protospacer adjacent motif (PAM) site. These mutations included single nucleotide substitutions leading to either amino acid changes or silent mutations. Notably, one of the detected mutations resulted in a T-to-G transition, changing a codon encoding phenylalanine to serine, while another induced a thymine-to-adenine transition, modifying the encoded tyrosine. A third mutation occurred but did not alter the amino acid sequence. All mutations were located in close proximity to the PAM site, which is consistent with known CRISPR/Cas9 cleavage patterns.
 
Discussion
This study represents the first successful application of CRISPR/Cas9-mediated mutagenesis of the CenH3 gene in cucumber. The induced mutations, although not immediately lethal, demonstrate the potential of CenH3 as a target for haploid induction. Prior studies in other species, such as carrot and Arabidopsis, have shown that mutations in CenH3 can lead to genome elimination and the generation of haploid progeny. However, the high resistance of cucumber to haploid induction—possibly due to the gene structure and the scarcity of optimal PAM sites—presents a significant challenge. The limited number of targetable sites near the functional domain of CenH3 in cucumber contrasts with more flexible systems in crops like carrot, where diverse guide RNAs can be designed for efficient cleavage. This underscores the importance of identifying alternative CRISPR systems or engineering novel PAM recognition variants to enhance editing efficiency in cucurbits. Despite the technical success in generating targeted CenH3 mutations, the study could not confirm haploid induction in the transformed cucumber plants. Nevertheless, the results open new avenues for future research into optimizing transformation systems and testing the phenotypic consequences of CenH3 disruption in subsequent generations.
 
Conclusion
This research provides a proof-of-concept for CRISPR/Cas9-mediated editing of the CenH3 gene in cucumber, laying the groundwork for potential development of haploid inducer lines in Cucurbitaceae. The successful induction of three mutations in the target gene suggests that, with further optimization, this strategy may be harnessed to accelerate cucumber breeding programs. Given the challenges in achieving effective PAM positioning and the essential role of CenH3 in cell viability, alternative CRISPR systems or multiplexed editing approaches may be necessary to enhance the haploid induction efficiency in this recalcitrant species.
 
Acknowledgements
This research was conducted under the financial and moral support of the Agricultural Biotechnology Laboratory, Plant Production and Genetics Department, Faculty of Agriculture, Bu-Ali Sina University.