Animal Bioscience

  • 제38권3호
  • /
  • Pages.560-567
  • /
  • 2025
  • /
  • 2765-0189(pISSN)
  • /
  • 2765-0235(eISSN)
아세아태평양축산학회 (Asian Australasian Association of Animal Production Societies)
권호 표지
DOI QR코드

DOI QR Code

The decrease in Rad51 and DNA ligase IV nuclear protein expression in Msh2 knockdown HC11 cells induced the low CRISPR/Cas9-mediated knock-in efficiency at the β-casein gene locus

  • Ga-Yeon Kim (Department of Animal Science, Chonnam National University) ;
  • Man-Jong Kang (Department of Animal Science, Chonnam National University)
  • 투고 : 2024.04.04
  • 심사 : 2024.09.03
  • 발행 : 2025.03.01
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

Objective: Successful gene editing technology is crucial in molecular biology and related fields. An essential part of an efficient knock-in system is increasing homologous recombination (HR) efficiency in the double-strand break (DSB) repair pathways. Interestingly, HR is closely related to the DNA mismatch repair (MMR) pathway, whereby MMR-related gene Msh2 recognizes a mismatch of nucleotides in recombinant intermediates or gene conversion formed during HR. This study aimed to investigate how the knockdown of Msh2 affects HR-mediated knock-in efficiency at the mouse β-casein locus. Therefore, we investigated the effect of inhibiting Msh2 expression on the expression of the HR-related gene Rad51 and the key enzyme DNA ligase IV involved in non-homologous end joining (NHEJ). Methods: The knock-in vector targeting the mouse β-casein gene locus, programmed guide RNA, and Msh2 siRNA expression vector were co-transfected in HC11 cells, or only the Msh2 siRNA expression vector was transfected. Knock-in efficiency was confirmed by polymerase chain reaction (PCR). The mRNA and protein expression of Msh2, HR-related gene Rad51, and NHEJ-related gene DNA ligase IV were evaluated by quantitative reverse transcription PCR (RT-qPCR) and Western blot analysis. Results: The knock-in vector efficiency at the mouse β-casein gene locus significantly decreased upon Msh2 knockdown in HC11 mouse mammary epithelial cells (HC11 cell). Additionally, the knockdown of the DNA MMR-related gene Msh2 protein significantly downregulated the nuclear protein expression of the HR-related Rad51 and NHEJ-related DNA ligase IV genes. Conclusion: The decreased Msh2 protein expression in the nucleus downregulated the Rad51 and ligase IV protein expressions. Consequently, reduced Rad51 expression results in decreased knock-in efficiency.

키워드

과제정보

This work was supported by a National Research Foundation of Korea (NRF) grant awarded by the Korean government (MSIT) (No.2021R1F1A1052105 and RS-2024-00343478).

참고문헌

  1. Li T, Yang Y, Qi H, et al. CRISPR/Cas9 therapeutics: progress and prospects. Signal Transduct Target Ther 2023;8:36. https://doi.org/10.1038/s41392-023-01309-7
  2. Gaj T, Sirk SJ, Shui SL, Liu J. Genome-editing technologies: principles and applications. Cold Spring Harb Perspect Biol 2016;8:a023754. https://doi.org/10.1101/cshperspect.a023754
  3. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 2012;337:816-21. https://doi.org/10.1126/science.1225829
  4. Vartak SV, Raghavan SC. Inhibition of nonhomologous end joining to increase the specificity of CRISPR/Cas9 genome editing. FEBS J 2015;282:4289-94. https://doi.org/10.1111/febs.13416
  5. Frankenberg-Schwager M, Gebauer A, Koppe C, Wolf H, Pralle E, Frankenberg D. Single-strand annealing, conservative homologous recombination, nonhomologous DNA end joining, and the cell cycle-dependent repair of DNA double-strand breaks induced by sparsely or densely ionizing radiation. Radiat Res 2009;171:265-73. https://doi.org/10.1667/RR0784.1
  6. Sander JD, Joung JK. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol 2014;32:347-5. https://doi.org/10.1038/nbt.2842
  7. Mao Z, Bozzella M, Seluanov A, Gorbunova V. DNA repair by nonhomologous end joining and homologous recombination during cell cycle in human cells. Cell Cycle 2008;7:2902-6. https://doi.org/10.4161/cc.7.18.6679
  8. Chu VT, Weber T, Wefers B, et al. Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat Biotechnol 2015;33:543-8. https://doi.org/10.1038/nbt.3198
  9. Maruyama T, Dougan SK, Truttmann MC, Bilate AM, Ingram JR, Ploegh HL. Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nat Biotechnol 2015;33:538-42. https://doi.org/10.1038/nbt.3190
  10. Arnaudeau C, Helleday T, Jenssen D. The RAD51 protein supports homologous recombination by an exchange mechanism in mammalian cells. J Mol Biol 1999;289:1231-8. https://doi.org/10.1006/jmbi.1999.2856
  11. Park SJ, Yoon S, Choi EH, Hyeon H, Lee K, Kim KP. Elevated expression of exogenous RAD51 enhances the CRISPR/ Cas9-mediated genome editing efficiency. BMB Rep 2023;56: 102-7. https://doi.org/10.5483/BMBRep.2022-0149
  12. Banan M. Recent advances in CRISPR/Cas9-mediated knock-ins in mammalian cells. J Biotechnol 2020;308;1-9. https://doi.org/10.1016/j.jbiotec.2019.11.010
  13. Abe M, Nakatsukasa E, Natsume R, et al. A novel technique for large-fragment knock-in animal production without ex vivo handling of zygotes. Sci Rep 2023;13:2245. https://doi.org/10.1038/s41598-023-29468-1
  14. Erwood S, Gu B. Embryo-based large fragment knock-in in mammals: why, how and what's next. Genes 2020;11:140. https://doi.org/10.3390/genes11020140
  15. Spell RM, Jinks-Robertson S. Role of mismatch repair in the fidelity of RAD51- and RAD59-dependent recombination in Saccharomyces cerevisiae. Genetics 2003;165:1733-44. https://doi.org/10.1093/genetics/165.4.1733
  16. Worrillow LJ, Allan JM. Deregulation of homologous recombination DNA repair in alkylating agent-treated stem cell clones: a possible role in the aetiology of chemotherapy-induced leukaemia. Oncogene 2006;25:1709-20. https://doi.org/10.1038/sj.onc.1209208
  17. Parekh-Olmedo H, Ferrara L, Brachman E, Kmiec EB. Gene therapy progress and prospects: targeted gene repair. Gene Ther 2005;12:639-46. https://doi.org/10.1038/sj.gt.3302511
  18. Spies M, Fishel R. Mismatch repair during homologous and homeologous recombination. Cold Spring Harb Perspect Biol 2015;7:a022657. https://doi.org/10.1101/cshperspect.a022657
  19. Kim SY, Kim GY, You HJ, Kang MJ. Relationship between DNA mismatch repair and CRISPR/Cas9-mediated knockin in the bovine β-casein gene locus. Anim Biosci 2022;35:126-37. https://doi.org/10.5713/ab.21.0117
  20. Kim GY, Kang MJ. Cadmium chloride down-regulates the expression of Rad51 in HC11 cells and reduces knock-in efficiency. J Anim Reprod Biotechnol 2023;38:99-108. https://doi.org/10.12750/JARB.38.3.99
  21. Ceccaldi R, Rondinelli B, D'Andrea AD. Repair pathway choices and consequences at the double-strand break. Trends Cell Biol 2016;26:52-64. https://doi.org/10.1016/j.tcb.2015.07.009
  22. Liu D, Keijzers G, Rasmussen LJ. DNA mismatch repair and its many roles in eukaryotic cells. Mutat Res Rev Mutat Res 2017;773:174-87. https://doi.org/10.1016/j.mrrev.2017.07.001
  23. Harfe BD, Jinks-Robertson S. DNA mismatch repair and genetic instability. Annu Rev Genet 2000;34:359-99. https://doi.org/10.1146/annurev.genet.34.1.359
  24. Evans E, Alani E. Roles for mismatch repair factors in regulating genetic recombination. Mol Cell Biol. 2000;20:7839-44. https://doi.org/10.1128/MCB.20.21.7839-7844.2000
  25. Li J, Baker MD. Formation and repair of heteroduplex DNA on both sides of the double-strand break during mammalian gene targeting. J Mol Biol 2000;295:505-16. https://doi.org/10.1006/jmbi.1999.3400
  26. Elliott B, Jasin M. Repair of double-strand breaks by homologous recombination in mismatch repair-defective mammalian cells. Mol Cell Biol 2001;21:2671-82. https://doi.org/10.1128/MCB.21.8.2671-2682.2001
  27. Barwell J, Pangon L, Hodgson S, et al. Biallelic mutation of MSH2 in primary human cells is associated with sensitivity to irradiation and altered RAD51 foci kinetics. J Med Genet 2007;44:516-20. https://doi.org/10.1136/jmg.2006.048660
  28. Villemure JF, Abaji C, Cousineau I, Belmaaza A. MSH2-deficient human cells exhibit a defect in the accurate termination of homology-directed repair of DNA double-strand breaks. Cancer Res 2003;63:3334-9.
  29. Lin CT, Lyu YL, Xiao H, Lin WH, Whang-Peng J. Suppression of gene amplification and chromosomal DNA integration by the DNA mismatch repair system. Nucleic Acids Res 2001;29:3304-10. https://doi.org/10.1093/nar/29.16.3304
  30. Sible E, Attaway M, Fiorica G, Michel G, Chaudhuri J, Vuong BQ. Ataxia telangiectasia mutated and MSH2 control blunt DNA end joining in Ig class switch recombination. J Immunol 2023;210:369-76. https://doi.org/10.4049/jimmunol.2200590