Cas protein diagnostics for pathogen nucleic acids

Cas protein diagnostics for pathogen nucleic acids - Akashaditya Das I formulated my research goals for my PhD in tandem with a Global Challenges Research Grant awarded to the Ajioka Lab in collaboration with other labs in the United Kingdom and South Africa. The grant aimed to develop tests for pathogens that avoid the reliance on expensive equipment such as thermocyclers to function. We identified Cas proteins as the core technology to develop further and split ourselves into two teams - one focusing on a DNA target and one focusing on an RNA target. The Ajioka Lab was assigned to create a test for a DNA target and we selected Hepatitis B Virus to be our test case. In my thesis, I investigate the process for producing tests using CRISPR Cas technology for detecting the Hepatitis B Virus. I develop the tests with fluorescent readout and show how they can be adapted to work with lateral flow technology. I looked at integrating isothermal amplification technologies into the assay workflow to boost the test sensitivity. I looked at creating a one-pot system combining CRISPR Cas and isothermal amplification technologies. I also focus on developing semi-automated workows to enable higher throughput and improve the ability to screen multiple assay designs. I use the data from this workflow to develop a predictive tool using machine learning techniques that will allow researchers to identify the effectiveness of different CRISPR Cas assay designs in silico.