Animal Bioscience

Asian Australasian Association of Animal Production Societies (아세아태평양축산학회)
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Relationship between DNA mismatch repair and CRISPR/Cas9-mediated knock-in in the bovine β-casein gene locus

  • Kim, Seung-Yeon (Department of Animal Science, College of Agriculture and Life Science, Chonnam National University) ;
  • Kim, Ga-Yeon (Department of Animal Science, College of Agriculture and Life Science, Chonnam National University) ;
  • You, Hyeong-Ju (Department of Animal Science, College of Agriculture and Life Science, Chonnam National University) ;
  • Kang, Man-Jong (Department of Animal Science, College of Agriculture and Life Science, Chonnam National University)
  • Received : 2021.03.16
  • Accepted : 2021.06.16
  • Published : 2022.01.01
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Abstract

Objective: Efficient gene editing technology is critical for successful knock-in in domestic animals. RAD51 recombinase (RAD51) gene plays an important role in strand invasion during homologous recombination (HR) in mammals, and is regulated by checkpoint kinase 1 (CHK1) and CHK2 genes, which are upstream elements of RAD51 recombinase (RAD51). In addition, mismatch repair (MMR) system is inextricably linked to HR-related pathways and regulates HR via heteroduplex rejection. Thus, the aim of this study was to investigate whether clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9)-mediated knock-in efficiency of human lactoferrin (hLF) knock-in vector in the bovine β-casein gene locus can be increased by suppressing DNA MMR-related genes (MSH2, MSH3, MSH6, MLH1, and PMS2) and overexpressing DNA double-strand break (DSB) repair-related genes (RAD51, CHK1, CHK2). Methods: Bovine mammary epithelial (MAC-T) cells were transfected with a knock-in vector, RAD51, CHK1, or CHK2 overexpression vector and CRISPR/sgRNA expression vector to target the bovine β-casein gene locus, followed by treatment of the cells with CdCl2 for 24 hours. After 3 days of CdCl2 treatment, the knock-in efficiency was confirmed by polymerase chain reaction (PCR). The mRNA expression levels of DNA MMR-related and DNA DSB repair-related genes were assessed by quantitative real-time PCR (RT-qPCR). Results: Treatment with CdCl2 decreased the mRNA expression of RAD51 and MMRrelated genes but did not increase the knock-in efficiency in MAC-T cells. Also, the overexpression of DNA DSB repair-related genes in MAC-T cells did not significantly affect the mRNA expression of MMR-related genes and failed to increase the knock-in efficiency. Conclusion: Treatment with CdCl2 inhibited the mRNA levels of RAD51 and DNA MMR-related genes in MAC-T cells. However, the function of MMR pathway in relation to HR may differ in various cell types or species.

Keywords

Acknowledgement

This work was supported by a National Research Foundation of Korea (NRF) grant awarded by the Korea government (MSIT) (No. 2019R1F1A1061424) and the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through the Agri-Bio Industry Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (No. 316037-4).

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