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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 Kim1 
, Man-Jong Kang1,*
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Department of Animal Science, Chonnam National University, Gwangju, Korea |
Correspondence:
Man-Jong Kang, Tel: +82-62-530-2113, Fax: +82-62-530-2129, Email: mjkang@jnu.ac.kr
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Received: 4 April 2024 • Revised: 28 July 2024 • Accepted: 3 September 2024 |
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| Abstract |
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.
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| Keywords:
Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 9 (CRISPR/Cas9); DNA Mismatch Repair (MMR); Homologous Recombination (HR); Non-homologous End Joining (NHEJ) |
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