Exploration of the influence of KHDC3L gene knock-out by CRISPR/Cas9 technology on PEG3 promoter

Document Type : Original Article

Authors

1 Department of Medical Biotechnology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran

2 Epilepsy Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

3 Assistant Professor of Periodontics, Shiraz University of Medical Sciences, Shiraz, Iran

4 Department of Genetics, Faculty of Advanced Science and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran

5 Faculty of Interdisciplinary Science and Technology, Tarbiat Modares University, Tehran, Iran

6 Department of Microbiology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

7 Infertility Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

10.30476/tips.2023.99754.1208

Abstract

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.

Keywords

References

1.    Khalili Alashti S, Fallahi J, Jokar A, Fardaei M. CRISPR/Cas9 knock-in toward creating a Rett syndrome cell model with a synonymous mutation in the MECP2 gene. J Gene Med. 2020 Nov;22(11):e3258. doi: 10.1002/jgm.3258. Epub 2020 Aug 19. PMID: 32761967.
2.    Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013 Nov;8(11):2281-2308. doi: 10.1038/nprot.2013.143. Epub 2013 Oct 24. PMID: 24157548; PMCID: PMC3969860.
3.    Doudna JA, Charpentier E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science. 2014 Nov 28;346(6213):1258096. doi: 10.1126/science.1258096. PMID: 25430774.
4.    Redman M, King A, Watson C, King D. What is CRISPR/Cas9? Arch Dis Child Educ Pract Ed. 2016 Aug;101(4):213-5. doi: 10.1136/archdischild-2016-310459. Epub 2016 Apr 8. PMID: 27059283; PMCID: PMC4975809.
5.    Jiang F, Doudna JA. CRISPR-Cas9 Structures and Mechanisms. Annu Rev Biophys. 2017 May 22;46:505-529. doi: 10.1146/annurev-biophys-062215-010822. Epub 2017 Mar 30. PMID: 28375731.
6.    Fallahi J, Alashti SK, Aliabadi BE, Mohammadi S, Fardaei M. Recurrent pregnancy loss in the female with a heterozygous mutation in KHDC3L gene. Gene Rep. 2020;20:100721.
7.    Zhang W, Chen Z, Zhang D, Zhao B, Liu L, Xie Z, et al. KHDC3L mutation causes recurrent pregnancy loss by inducing genomic instability of human early embryonic cells. PLoS Biol. 2019 Oct 14;17(10):e3000468. doi: 10.1371/journal.pbio.3000468. PMID: 31609975; PMCID: PMC6812846.
8.    Demond H, Anvar Z, Jahromi BN, Sparago A, Verma A, Davari M, et al. A KHDC3L mutation resulting in recurrent hydatidiform mole causes genome-wide DNA methylation loss in oocytes and persistent imprinting defects post-fertilisation. Genome Med. 2019;11(1):1-14.
9.    Li L, Keverne EB, Aparicio SA, Ishino F, Barton SC, Surani MA. Regulation of maternal behavior and offspring growth by paternally expressed Peg3. Science. 1999 Apr 9;284(5412):330-3. doi: 10.1126/science.284.5412.330. PMID: 10195900.
10.    Kuroiwa Y, Kaneko-Ishino T, Kagitani F, Kohda T, Li LL, Tada M, et al. Peg3 imprinted gene on proximal chromosome 7 encodes for a zinc finger protein. Nat Genet. 1996 Feb;12(2):186-90. doi: 10.1038/ng0296-186. PMID: 8563758.
11.    He H, Kim J. Regulation and function of the peg3 imprinted domain. Genomics Inform. 2014 Sep;12(3):105-13. doi: 10.5808/GI.2014.12.3.105. Epub 2014 Sep 30. PMID: 25317109; PMCID: PMC4196374.
12.    Licchesi JD, Herman JG. Methylation-specific PCR. Methods Mol Biol. 2009;507:305-23. doi: 10.1007/978-1-59745-522-0_22. PMID: 18987823.
13.    Sanchez-Delgado M, Martin-Trujillo A, Tayama C, Vidal E, Esteller M, Iglesias-Platas I, et al. Absence of Maternal Methylation in Biparental Hydatidiform Moles from Women with NLRP7 Maternal-Effect Mutations Reveals Widespread Placenta-Specific Imprinting. PLoS Genet. 2015 Nov 6;11(11):e1005644. doi: 10.1371/journal.pgen.1005644. PMID: 26544189; PMCID: PMC4636177.
14.    Hui P, Buza N, Murphy KM, Ronnett BM. Hydatidiform Moles: Genetic Basis and Precision Diagnosis. Annu Rev Pathol. 2017 Jan 24;12:449-485. doi: 10.1146/annurev-pathol-052016-100237. PMID: 28135560.
15.    Ito Y, Maehara K, Kaneki E, Matsuoka K, Sugahara N, Miyata T, et al. Novel Nonsense Mutation in the NLRP7 Gene Associated with Recurrent Hydatidiform Mole. Gynecol Obstet Invest. 2016;81(4):353-8. doi: 10.1159/000441780. Epub 2015 Nov 26. PMID: 26606510.
16.    Akoury E. Role of the nucleotide oligomerization domain-like receptor protein 7 in the pathology of recurrent hydatidiform moles: McGill University (Canada); 2016;12(2):22-31..
17.    Hidaka H, Higashimoto K, Aoki S, Mishima H, Hayashida C, Maeda T, et al. Comprehensive methylation analysis of imprinting-associated differentially methylated regions in colorectal cancer. Clin Epigenetics. 2018 Dec 4;10(1):150. doi: 10.1186/s13148-018-0578-9. PMID: 30509319; PMCID: PMC6278095.
18.    Sazhenova E, Nikitina T, Skryabin N, Minaycheva L, Ivanova T, Nemtseva T, et al. Epigenetic status of imprinted genes in placenta during recurrent pregnancy loss. Russ J Genet. 2017;53:376-87.
19.    Moein-Vaziri N, Fallahi J, Namavar-Jahromi B, Fardaei M, Momtahan M, Anvar Z. Clinical and genetic-epignetic aspects of recurrent hydatidiform mole: A review of literature. Taiwan J Obstet Gynecol. 2018 Feb;57(1):1-6. doi: 10.1016/j.tjog.2017.12.001. PMID: 29458875.
20.    El-Maarri O, Seoud M, Coullin P, Herbiniaux U, Oldenburg J, Rouleau G, et al. Maternal alleles acquiring paternal methylation patterns in biparental complete hydatidiform moles. Hum Mol Genet. 2003 Jun 15;12(12):1405-13. doi: 10.1093/hmg/ddg152. PMID: 12783848.
21.    Kou YC, Shao L, Peng HH, Rosetta R, del Gaudio D, Wagner AF, et al. A recurrent intragenic genomic duplication, other novel mutations in NLRP7 and imprinting defects in recurrent biparental hydatidiform moles. Mol Hum Reprod. 2008 Jan;14(1):33-40. doi: 10.1093/molehr/gam079. Epub 2007 Nov 26. PMID: 18039680.
22.    Monk D, Sanchez-Delgado M, Fisher R. NLRPs, the subcortical maternal complex and genomic imprinting. Reproduction. 2017 Dec;154(6):R161-R170. doi: 10.1530/REP-17-0465. Epub 2017 Sep 15. PMID: 28916717.
23.    Aghajanova L, Mahadevan S, Altmäe S, Stavreus-Evers A, Regan L, Sebire N, et al. No evidence for mutations in NLRP7, NLRP2 or KHDC3L in women with unexplained recurrent pregnancy loss or infertility. Hum Reprod. 2015 Jan;30(1):232-8. doi: 10.1093/humrep/deu296. Epub 2014 Nov 5. PMID: 25376457; PMCID: PMC4262469.
Trends in Pharmaceutical Sciences
Volume 9, Issue 4
December 2023
Pages 253-260

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