As a first step, all of the necessary components of the PaeCas3c system were moved into a laboratory model host bacterium for easier study. The system was reprogrammed to target the DNA of this model bacterium itself (so that it is targeting the host bacteria's genome) and resulted in large deletions at the intended target DNA. Through multiple rounds of design and testing, we were able to modify the system so that it was optimized to achieve editing efficiencies of over 90 %. Subsequently, as a proof-of-principle to demonstrate the capabilities of the PaeCas3c system for gene editing, we targeted several different regions of the host bacteria's genome to create strains with multiple deletions towards the goal of creating a minimal genome strain. We rapidly created a strain with 16 distinct deletions with ~20 % of its original genome missing. Overall, this clearly demonstrates the potential for PaeCas3c as a tool to make rapid, large modifications for strain engineering purposes. Importantly, a direct comparison to the state-of-the-art CRISPR-Cas9-based technique showed that this scale of modification was only achievable using PaeCas3c.
A next hurdle to overcome was to adapt PaeCas3c to other organisms. In order to achieve this, we moved all of the necessary components of the system onto a single plasmid, a mobilizable DNA element used to transfer genetic information from one bacteria to another. After several rounds of optimization, we were able to demonstrate that the system is capable of creating large genomic deletions in a variety of bacterial organisms, including E. coli, P. syringae, and K. pneumoniae. Overall, this clearly demonstrates the potential for PaeCas3c to be a truly universal gene-editing tool, just like CRISPR-Cas9 systems.
Once this tool was available, we applied it to study interactions between clinical bacterial strains and wide panels of bacteriophages, viruses that infect bacteria. Testing of strains with large deletions have allowed us to identify two novel factors involved in bacterial-phage interactions in various strains of P. syringae, a ferrichrome receptor required for the infection of a range of different phages, as well as a novel type IIS restriction enzyme that targets a wide variety of different bacteriophages.
Furthermore, we have been able to successfully adapt this editing system to be functional in HEK293T human cell lines, generating deletions larger relative to CRISPR-Cas9 technology. Although further characterization of these events are required, this clearly demonstrates the far-reaching potential of these systems.
I have been continuously disseminating our results through multiple forums, primarily in the form of openly accessible publications (2 total so far funded by this project, with another to be submitted in the near-future), as well as at multiple scientific conferences as both poster and oral presentations.
