Cloning, Expression, and Functional Analysis of the Compact Anoxybacillus flavithermus Cas9 Nuclease

Research on Cas9 nucleases from different organisms holds great promise for advancing genome engineering and gene therapy tools as it could provide novel structural insights into CRISPR editing mechanisms, expanding its application area in biology and medicine. The current study focuses on generating a construct to express a compact Cas9 nuclease (AnoCas9) from the thermophilic microorganism Anoxybacillus flavithermus. Next, distinctive AnoCas9 properties are investigated. AnoCas9 gene is expressed in E.coli producing a polypeptide fused with the maltose-binding protein (MBP-AnoCas9). His-Tag in its structure enables purification using metal-chelate chromatography followed by the cleavage of the polypeptide by TEV protease. Bioinformatical analysis of the CRISPR array found in the genome of an Anoxybacillus flavithermus strain helps predict a functional PAM sequence for AnoCas9, which is supported by in vitro experiments. The purified protein demonstrates nuclease activity in the presence of crRNA:tracrRNA duplex in the 37-60 °С range, with maximum activity observed at 45-55 °С. The analysis of FAM-labeled dsDNA substrate cleavage has allowed us to determine the functional AnoCas9 PAM motif as 5’-NNNNCDAA-3’. Thus, AnoCas9 adds to the repertoire of thermophilic Cas9 effectors and its properties suggest application in areas requiring the presence of thermostable CRISPR/Cas systems.