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

• Published in: Animal bioscience Vol. 38; no. 3; pp. 560 - 567
• Main Authors: Kim, Ga-Yeon, Kang, Man-Jong
• Format: Journal Article
• Language: English
• Published: Korea (South) Animal Bioscience 01.03.2025; Asian-Australasian Association of Animal Production Societies; 아세아·태평양축산학회
• Subjects: clustered regularly interspaced short palindromic repeats/crispr-associated 9 (crispr/cas9); dna mismatch repair (mmr); homologous recombination (hr); non-homologous end joining (nhej); 축산학; Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-Associated 9 (CRISPR/Cas9); DNA Mismatch Repair (MMR); Homologous Recombination (HR); Non-Homologous End Joining (NHEJ)
• ISSN: 2765-0189; 2765-0235
• DOI: 10.5713/ab.24.0206

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.