Most higher organisms have complex immune systems that help fight off viral infections. Bacteria have simpler immune mechanisms that seek and destroy viral DNA. They use enzymes and a specific 'guide' that help to identify and destroy the infecting virus DNA. The simplicity of these systems allow them to be re-purposed for a range of biotechnological applications. These specific 'guides' can recognize DNA even if it has only a partial match. This turns out to be a useful characteristic for bacteria but can be highly problematic for most biotechnological applications. This project has the overall goals of understanding how this feature benefits bacterial immunity and of improving the efficiency of tools based on this emerging technology. The PI will offer a new course for both science and non-science majors about the science and ethics of CRISPR-Cas technology. This course will help train students in science communication and to engage in informal scientific discussions with the public.
The overall goal of this proposal is to define and improve the sequence specificity of Class 2 CRISPR-Cas endonucleases. The specific scientific goals are to: 1) define the effects of gRNA and target sequence on the specificity of Cas endonucleases using high-throughput screens; 2) determine how Cas endonuclease specificity affects CRISPR-Cas immunity against rapidly evolving viral invaders; 3) create a complementary tool for improved Cas endonuclease specificity using chemically modified gRNAs. Successful completion of the proposed work will reveal the importance of CRISPR-Cas fidelity for endogenous immunity and host-virus co-evolution. In addition, development of high-throughput specificity screens will enable rapid assessment of a variety of Cas endonucleases that could be harnessed for biotechnological applications. Results from these screens will facilitate improved design of gRNAs to reduce the risk of non-specific genome editing. Finally, studies of chemically modified gRNAs will catalog the effects of chemical modifications on Cas endonuclease activity and specificity, allowing for the identification of optimal modification sites and implementation of these improved gRNAs in in vivo genome editing studies.