Exploration of the influence of KHDC۳L gene knock-out by CRISPR/Cas۹ technology on PEG۳ promoter

Background and aim: CRISPR-Cas۹, enable precise DNA manipulation via RNA-guided breaks. KHDC۳L and PEG۳ genes are vital; CRISPR-Cas۹ studies their roles in reproduction, development, and gene regulation. Focusing on KHDC۳L's impact on PEG۳ promoter in HCT۱۱۶ cells, insights into gene functions and disease mechanisms emerge, informing potential therapies. Method: This study aims to design sgRNAs for the KHDC۳L gene using CRISPR tools involved ranking, off-target evaluation, and cloning. HCT۱۱۶ cells were cultured, synchronized, and transfected with sgRNAs using lipofectamine. Successful transfections were confirmed by fluorescence microscopy. Clonal expansion followed, with DNA extracted and genotyped using PCR and Sanger sequencing. Bisulfite conversion analyzed DNA methylation, employing restriction enzymes for CpG site analysis. Statistical significance (p≤۰.۰۵) was assessed using SPSS software. Results: The neighboring regions exhibited significant genomic changes. The designed sgRNAs were cloned into the PX۴۵۸ plasmid, directing Cas۹ to create double-strand breaks (DSBs) in KHDC۳L exon ۳. Transfected cells showed around ۶۵% efficiency. Gap-PCR confirmed knock-out in ۳ out of ۱۷ clones. COBRA analysis revealed allele-specific CpG island methylation in PEG۳, indicating the impact of KHDC۳L knock-out on PEG۳ promoter methylation and expression. Conclusion: The study demonstrates increased PEG۳ promoter methylation upon KHDC۳L deletion, indicating its role in modulation. Knockout correlates with reduced cell proliferation and colony formation, suggesting KHDC۳L's role in promoting cell growth. The gene's relevance in PEG۳ regulation and potential therapeutic implications are underscored, though further mechanistic insights are warranted.

کلیدواژه ها:

KHDC۳L gene ، PEG۳ promoter ، CRISPR/Cas۹ ، gene knock-out