Relationship between DNA mismatch repair and CRISPR/Cas9-mediated knock-in in the bovine β-casein gene locus

Abstract

Objective: Efficient gene editing technology is critical for successful knock-in in domestic animals.RAD51 gene plays an important role in strand invasion during homologous recombination (HR) in mammals, and is regulated by CHK1 and CHK2 genes, which are upstream elements of 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 CRISPR/Cas9mediated knock-in efficiency of human lactoferrin (hLF) knock-in vector in the bovine β-casein gene locus can be increased by suppressing DNA mismatch repair (MMR)-related genes (MSH2, MSH3, MSH6, MLH1 and PMS2) and overexpressing DNA double-strand break (DSB) repair-related genes (RAD51, CHK1, CHK2). Methods: 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 CdCl 2 for 24 hours. After 3 days of CdCl 2 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 realtime PCR (RT-qPCR).Results: Treatment with CdCl 2 decreased the mRNA expression of RAD51 and MMR-related 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 MMRrelated genes and failed to increase the knock-in efficiency. Conclusion:Treatment with CdCl 2 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 A c c e p t e d A r t i c l e various cell types or species.

Cadmium chloride down-regulates the expression of Rad51 in HC11 cells and reduces knock-in efficiency

“…These reports suggest that the inhibition of MutS α and MutS β by CdCl 2 treatment may increase HR efficiency. However, it has been reported that low-dose CdCl 2 (1 μM) in Mac-T cells does not increase HR efficiency (Kim et al, 2022).…”

“…This suggests that these results may be attributed to the suppression of the MMR pathway initiator gene Msh2. A study byKim et al (2022) reported that low-dose CdCl 2 treatment does not inhibit MMR-related genes when CRISPR/Cas9-mediated gene targeting is performed at the bovine beta-casein locus. Moreover, failure to suppress the expression of MMR-related genes has been reported as a potential cause of reduced knock-in efficiency.In this study, high concentrations of cadmium were used to compensate for the problem of MMR-related gene inhibition.…”