Exploration of the influence of KHDC3L gene knock-out by CRISPR/Cas9 technology on PEG3 promoter
Background and aim: CRISPR-Cas9, enable precise DNA manipulation via RNA-guided breaks. KHDC3L and PEG3 genes are vital; CRISPR-Cas9 studies their roles in reproduction, development, and gene regulation. Focusing on KHDC3L's impact on PEG3 promoter in HCT116 cells, insights into gene functions...
| Main Authors: | , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Shiraz University of Medical Sciences
2023-12-01
|
| Series: | Trends in Pharmaceutical Sciences |
| Subjects: | |
| Online Access: | https://tips.sums.ac.ir/article_49730_569136271724191afa9f85fcec28bc47.pdf |
| _version_ | 1797216818004230144 |
|---|---|
| author | Arezoo Jokar Shayan Khalili Alashti Maryam Baneshi Ali Rastegarian Kimia Sadat Hashemi Mohadese Koohi Aliabadi Mojtaba Kashfi amir savardashtaki |
| author_facet | Arezoo Jokar Shayan Khalili Alashti Maryam Baneshi Ali Rastegarian Kimia Sadat Hashemi Mohadese Koohi Aliabadi Mojtaba Kashfi amir savardashtaki |
| author_sort | Arezoo Jokar |
| collection | DOAJ |
| description | Background and aim: CRISPR-Cas9, enable precise DNA manipulation via RNA-guided breaks. KHDC3L and PEG3 genes are vital; CRISPR-Cas9 studies their roles in reproduction, development, and gene regulation. Focusing on KHDC3L's impact on PEG3 promoter in HCT116 cells, insights into gene functions and disease mechanisms emerge, informing potential therapies. Method: This study aims to design sgRNAs for the KHDC3L gene using CRISPR tools involved ranking, off-target evaluation, and cloning. HCT116 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≤0.05) was assessed using SPSS software. Results: The neighboring regions exhibited significant genomic changes. The designed sgRNAs were cloned into the PX458 plasmid, directing Cas9 to create double-strand breaks (DSBs) in KHDC3L exon 3. Transfected cells showed around 65% efficiency. Gap-PCR confirmed knock-out in 3 out of 17 clones. COBRA analysis revealed allele-specific CpG island methylation in PEG3, indicating the impact of KHDC3L knock-out on PEG3 promoter methylation and expression. Conclusion: The study demonstrates increased PEG3 promoter methylation upon KHDC3L deletion, indicating its role in modulation. Knockout correlates with reduced cell proliferation and colony formation, suggesting KHDC3L's role in promoting cell growth. The gene's relevance in PEG3 regulation and potential therapeutic implications are underscored, though further mechanistic insights are warranted. |
| first_indexed | 2024-04-24T11:52:00Z |
| format | Article |
| id | doaj.art-2669d064cd004cdb8767611ba5b8ce46 |
| institution | Directory Open Access Journal |
| issn | 2423-5652 |
| language | English |
| last_indexed | 2024-04-24T11:52:00Z |
| publishDate | 2023-12-01 |
| publisher | Shiraz University of Medical Sciences |
| record_format | Article |
| series | Trends in Pharmaceutical Sciences |
| spelling | doaj.art-2669d064cd004cdb8767611ba5b8ce462024-04-09T07:38:53ZengShiraz University of Medical SciencesTrends in Pharmaceutical Sciences2423-56522023-12-019425326010.30476/tips.2023.99754.120849730Exploration of the influence of KHDC3L gene knock-out by CRISPR/Cas9 technology on PEG3 promoterArezoo Jokar0Shayan Khalili Alashti1Maryam Baneshi2Ali Rastegarian3Kimia Sadat Hashemi4Mohadese Koohi Aliabadi5Mojtaba Kashfi6amir savardashtaki7Department of Medical Biotechnology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, IranDepartment of Medical Biotechnology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, IranDepartment of Medical Biotechnology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, IranAssistant Professor of Periodontics, Shiraz University of Medical Sciences, Shiraz, IranDepartment of Genetics, Faculty of Advanced Science and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, IranFaculty of Interdisciplinary Science and Technology, Tarbiat Modares University, Tehran, IranDepartment of Microbiology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, IranInfertility Research Center, Shiraz University of Medical Sciences, Shiraz, IranBackground and aim: CRISPR-Cas9, enable precise DNA manipulation via RNA-guided breaks. KHDC3L and PEG3 genes are vital; CRISPR-Cas9 studies their roles in reproduction, development, and gene regulation. Focusing on KHDC3L's impact on PEG3 promoter in HCT116 cells, insights into gene functions and disease mechanisms emerge, informing potential therapies. Method: This study aims to design sgRNAs for the KHDC3L gene using CRISPR tools involved ranking, off-target evaluation, and cloning. HCT116 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≤0.05) was assessed using SPSS software. Results: The neighboring regions exhibited significant genomic changes. The designed sgRNAs were cloned into the PX458 plasmid, directing Cas9 to create double-strand breaks (DSBs) in KHDC3L exon 3. Transfected cells showed around 65% efficiency. Gap-PCR confirmed knock-out in 3 out of 17 clones. COBRA analysis revealed allele-specific CpG island methylation in PEG3, indicating the impact of KHDC3L knock-out on PEG3 promoter methylation and expression. Conclusion: The study demonstrates increased PEG3 promoter methylation upon KHDC3L deletion, indicating its role in modulation. Knockout correlates with reduced cell proliferation and colony formation, suggesting KHDC3L's role in promoting cell growth. The gene's relevance in PEG3 regulation and potential therapeutic implications are underscored, though further mechanistic insights are warranted.https://tips.sums.ac.ir/article_49730_569136271724191afa9f85fcec28bc47.pdfkhdc3l genepeg3 promotercrispr/cas9gene knock-out |
| spellingShingle | Arezoo Jokar Shayan Khalili Alashti Maryam Baneshi Ali Rastegarian Kimia Sadat Hashemi Mohadese Koohi Aliabadi Mojtaba Kashfi amir savardashtaki Exploration of the influence of KHDC3L gene knock-out by CRISPR/Cas9 technology on PEG3 promoter Trends in Pharmaceutical Sciences khdc3l gene peg3 promoter crispr/cas9 gene knock-out |
| title | Exploration of the influence of KHDC3L gene knock-out by CRISPR/Cas9 technology on PEG3 promoter |
| title_full | Exploration of the influence of KHDC3L gene knock-out by CRISPR/Cas9 technology on PEG3 promoter |
| title_fullStr | Exploration of the influence of KHDC3L gene knock-out by CRISPR/Cas9 technology on PEG3 promoter |
| title_full_unstemmed | Exploration of the influence of KHDC3L gene knock-out by CRISPR/Cas9 technology on PEG3 promoter |
| title_short | Exploration of the influence of KHDC3L gene knock-out by CRISPR/Cas9 technology on PEG3 promoter |
| title_sort | exploration of the influence of khdc3l gene knock out by crispr cas9 technology on peg3 promoter |
| topic | khdc3l gene peg3 promoter crispr/cas9 gene knock-out |
| url | https://tips.sums.ac.ir/article_49730_569136271724191afa9f85fcec28bc47.pdf |
| work_keys_str_mv | AT arezoojokar explorationoftheinfluenceofkhdc3lgeneknockoutbycrisprcas9technologyonpeg3promoter AT shayankhalilialashti explorationoftheinfluenceofkhdc3lgeneknockoutbycrisprcas9technologyonpeg3promoter AT maryambaneshi explorationoftheinfluenceofkhdc3lgeneknockoutbycrisprcas9technologyonpeg3promoter AT alirastegarian explorationoftheinfluenceofkhdc3lgeneknockoutbycrisprcas9technologyonpeg3promoter AT kimiasadathashemi explorationoftheinfluenceofkhdc3lgeneknockoutbycrisprcas9technologyonpeg3promoter AT mohadesekoohialiabadi explorationoftheinfluenceofkhdc3lgeneknockoutbycrisprcas9technologyonpeg3promoter AT mojtabakashfi explorationoftheinfluenceofkhdc3lgeneknockoutbycrisprcas9technologyonpeg3promoter AT amirsavardashtaki explorationoftheinfluenceofkhdc3lgeneknockoutbycrisprcas9technologyonpeg3promoter |