Type 2 CRISPR/Cas Identification of Geobacillus thermoleovorans from Batu Kuwung Banten: In Silico Studies

The CRISPR-Cas system is a mechanism of bacterial immunity against viral infections, which is utilized as a genetic and diagnostic engineering tool. CRISPR-Cas Class 2 is part of the CRISPR-Cas system, which has a simple mechanism of action because it only requires one type of protein to edit DNA or RNA. In general, the Class 2 Cas protein used is derived from mesophilic bacteria. This causes the use of Cas Class 2 proteins (such as Cas9, Cas12, and Cas13) to be still limited by the reaction temperature, stability, and the possibility of high denaturation. Thermophilic bacteria provide an interesting solution because of their thermostable protein, which is still active at high temperatures. Previously, we identified Geobacillus thermoleovorans from Batu Kuwung, Banten, Indonesia. Therefore, in this study, we aim to do an in silico study to analyze type 2 CRISPR-Cas protein in thermophilic bacteria as an initial study using G. thermoleovorans as the preference of thermophilic bacteria. To do so, sequence data of existing mesophilic and thermophilic type 2 CRISPR-Cas were obtained from GENBANK were used. BLASTP tools were used to determine the similarity of type 2 CRISPR/Cas (Cas9, Cas12, and Cas13). The result shows that G. thermoleovorans only has the Cas9 gene without Cas12 and Cas13. Interestingly, Cas9 from thermophilic bacteria has a shorter size comparing to existing mesophilic Cas9. The frequency of amino acid analysis shows that arginine (Arg) has more occurrence among other types of amino acid in thermophilic Cas9 compared to mesophilic Cas9. It suggests that the thermostability of thermophilic Cas9 may be due to the high frequency of Arg amino acid.