BackgroundThe CRISPR-Cas system is an adaptive immune mechanism for bacteria and archaea to resist foreign invasion. Currently, Cas9 and Cpf1 have been widely studied and applied in gene editing. C2c1 is a newly discovered CRISPR-Cas system endonuclease. It has broad application prospects due to its small molecular weight and high substrate recognition specificity.ObjectivesBacillus thermoamylovoransC2c1(BthC2c1) was expressed inE. coliC43 (DE3) competent cells, purified, and the BthC2c1-sgRNA-dsDNA complex was assembled. The effect of temperature on the cleavage ability of the BthC2c1 system was investigated.MethodsThe cDNA of BthC2c1 was cloned into the vector pGEX-6P-1. BthC2c1 was expressed inE. coliC43(DE3) cells and purified using a GST affinity column and FPLC. The sgRNAs were transcribed and purifiedin vitro, and the complexes were assembled by gel filtration chromatography. The enzyme cleavage activity of BthC2c1 at different temperatures was investigated using anin vitrocleavage assay. Microscale Thermophoresis detected the affinity of the BthC2c1-sgRNA complexes to substrate DNA.ResultsBthC2c1 proteins were prokaryotically expressed and purified. The complex of BthC2c1 with sgRNA and dsDNA was assembled.In vitrocleavage assay results showed that BthC2c1 cleaved the target DNA at temperatures ranging from 37°C to 67°C. The cleavage ability of BthC2c1 at 42°C was stronger than that at 37°C. The results of affinity detection showed that the affinity between the BthC2c1-sgRNA complex and ds36/36 at 42°C was stronger than that at 37°C.ConclusionIn this study, BthC2c1 was expressed, purified, and assembled into a complex with sgRNA and dsDNA. BthC2c1 cleaved DNA within the temperature range of 37°C to 67°C. The affinity of BthC2c1-sgRNA to DNA at 42°C was significantly enhanced than that at 37°C. It may be related to its stringent substrate recognition pattern, which differs from Cas9 and Cpf1. The temperature-dependent affinity changes of substrate binding may be part of the reason for the stronger cleavage activity of BthC2c1 at 42°C. This study may provide an experimental basis for optimizing and modifying the C2c1 gene editing system.