This work has demonstrated the extremely effective operation of this platform at temperatures as low as 4oC, making it feasible to modify the genes in creatures, plants, or crop types that are sensitive to temperature.
The prokaryotic genomes of bacteria and other prokaryotic organisms include short DNA sequences called CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), which serve as a recall of prior bacteriophage (virus) attacks that the bacterium successfully repelled.
These flags are used by the Cas9 enzyme, a component of the bacterial defense system, to accurately identify and cut any foreign DNA, defending the bacterium from further attacks by bacteriophages with similar characteristics. The foundation of CRISPR-Cas9 technology, which has recently been proved in the editing of genes in cells and creatures, is the extraordinary accuracy of targeting the DNA sequences and then successfully cutting them.
Successful applications of CRISPR-Cas9 technology include fundamental investigations of gene function, agriculture, and medicine to improve our understanding of disease processes and their possible future therapeutics. Until now, 37 °C has traditionally been the temperature used for binding testing.
The Raman Research Institute (RRI), an autonomous institute of the Department of Science and Technology (DST), has investigated temperature-dependent binding and release of cleaved products by the Cas9 enzyme as a further step to advance this platform into the forefront of biomedical and analytical biotechnology.
Under the direction of Dr. Gautam V. Soni, Serene Rose David, Sumanth Kumar Maheshwaram, Divya Shet, and Mahesh B. Lakshminarayana have shown that the Cas9 enzymes strongly bind to the target at very low temperatures and remain bound to the cleaved DNA products even after the enzyme has completed its task. The bound products were then carefully freed using a chemical denaturant or high temperature (that makes proteins and DNA lose their 3-dimensional structure and become non-functional).
The previously unknown temperature range that might be suitable for the long-term preservation of biological materials is now open to the Cas9-based genetic toolkit thanks to research published in the Nature Portfolio magazine Scientific Reports.
The ability to modify the genomes of the less studied species known as cryophiles, with an ideal development temperature of 15°C, is also made possible by their studies of the great effectiveness of Cas9 binding to target at very low temperatures. The findings on the mechanics of Cas9-DNA binding and release will be crucial for creating temperature-dependent CRISPR-Cas9 applications. Additionally, it develops a quantitative grasp of this enzyme system's process for product release.
