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.

Homologous recombination is a fundamental process that occurs in all species, from bacteria to humans (Vispé et al., 1998). Rad51 and Dmc1, the central homologous pairing and strand exchange proteins, are the main recombinases in the homologous recombination pathway (Hinch et al., 2020). Dmc1 is known to be expressed only in meiosis, whereas Rad51 has been reported to be expressed in both meiosis and non-meiosis (Brown and Bishop, 2014). In order to confirm whether Rad51 and HR are related, various studies have been carried out to induce artificial double strand breaks (DSBs) with UV irradiation and ionizing-radiation within cells (Elvers et al., 2011; Julien et al., 2013). It has been reported that the expression of Rad51 is increased during the S phase when irradiated with UV in human cells (Tashiro et al., 2000).The DNA repair pathway HR is also associated with mismatch repair (MMR) (Spies and Fishel, 2015). MMR is a highly preserved post-replication repair system that rec-

No abstract