Periodic Reporting for period 2 - GenDels (Development of a new CRISPR-Cas3-based tool for large genomic deletions)
Reporting period: 2021-07-01 to 2022-06-30
The ability to genetically modify the DNA of model organisms (gene editing) is crucial for understanding biological processes and has wide implications for human health. The recent advent of a variety of gene-editing technologies has greatly enhanced our capabilities in modifying DNA of interest. Primary among these, a revolutionary technology called CRISPR-Cas9 has enabled changing DNA at an unprecedented scale in a variety of organisms, leading to impactful biological findings in all areas of basic and applied research. Tools based on this technology are suitable for a variety of gene editing applications, including generation of small deletions and insertions in genes which can help decipher the function of individual genes. Utilization of CRISPR-Cas9 as a tool to generate larger deletions is however limited. The ability to generate large deletions in the DNA of a variety of organisms is advantageous for multiple reasons: 1) vast regions of DNA (so-called "dark regions") with unknown function exist in all species, deletions of which could help better understand their functions, 2) the genomes of bacteria could be streamlined so that they function better for biotechnology purposes, 3) generating deletions in harmful bacteria could reveal the way they make their hosts sick 4) a system capable of generating large deletions could also be used to selectively kill dangerous bacteria.
We have developed a technology based on an immune system called CRISPR-Cas3 that occurs naturally in some bacteria to overcome the lack of such a method. CRISPR-Cas3 systems are unique in that they not only cut DNA like other systems, but also loosen up its structure by unwinding it. They are then able to “chew” back this unwound DNA, leading to the destruction of long stretches of DNA. These properties have allowed CRISPR-Cas3 systems to generate large deletions when repurposed as tools. Previously, CRISPR-Cas3 systems had been overlooked as gene-editing tools, due to their relatively complicated architecture. We identified a more compact CRISPR-Cas3 system in a strain of the commonly studied bacterium Pseudomonas aeruginosa that consists of only 4 proteins that are needed to recognize and destroy targeted DNA sequences. This specific system, termed PaeCas3c, is ideal to repurpose as an editing tool, as it is less complex than other such systems. With this in mind, we achieved three broad goals: 1) we studied if and how PaeCas3c can make deletions in bacteria, 2) we developed the tool to be able to function in several different types of bacteria and 3) and we developed the system to be able to use it on more complex cells, such as model human cell lines.
We have developed a technology based on an immune system called CRISPR-Cas3 that occurs naturally in some bacteria to overcome the lack of such a method. CRISPR-Cas3 systems are unique in that they not only cut DNA like other systems, but also loosen up its structure by unwinding it. They are then able to “chew” back this unwound DNA, leading to the destruction of long stretches of DNA. These properties have allowed CRISPR-Cas3 systems to generate large deletions when repurposed as tools. Previously, CRISPR-Cas3 systems had been overlooked as gene-editing tools, due to their relatively complicated architecture. We identified a more compact CRISPR-Cas3 system in a strain of the commonly studied bacterium Pseudomonas aeruginosa that consists of only 4 proteins that are needed to recognize and destroy targeted DNA sequences. This specific system, termed PaeCas3c, is ideal to repurpose as an editing tool, as it is less complex than other such systems. With this in mind, we achieved three broad goals: 1) we studied if and how PaeCas3c can make deletions in bacteria, 2) we developed the tool to be able to function in several different types of bacteria and 3) and we developed the system to be able to use it on more complex cells, such as model human cell lines.
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.
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.
Because of the continuous DNA chopping activity of the Cas3 enzyme, PaeCas3c has the ability to overcome the limitation of other CRISPR-based techniques in creating large genomic deletions. We have shown that in bacteria, PaeCas3c has the ability to generate deletions of over 400 000 basepairs, at least two orders of magnitude larger than what is commonly observed using the previous state-of-the-art techniques. Utilization of PaeCas3c also introduces an entirely new capability to the genome engineering toolbox, the ability to generate sets of semi-random deletions. This means that the specific target sequence is defined, while the exact boundaries of the deletions are more random. We find that targeting DNA with PaeCas3c results in cuts that are most often repaired through short (4-14 basepairs) similarities already present in genomes. As short similarities are common across all genomes, the DNA cuts are rarely repaired at identical positions. Sequencing of 6 different strains with multiple deletions generated at the same time, had no two deletions with identical boundaries. This randomness means that gene editing with PaeCas3c results in lots of cells with different deletion sizes. These cells can then be tested for specific properties and cells showing traits of interest can be isolated, sequenced, and compared to other strains to identify the genomic sequence responsible for the trait. CRISPR-Cas3 editing is the first technology that allows this type of approach for testing and identifying potential genes of interest. We have employed the technology to identify previously unknown factors involved in interactions between clinical bacteria and bacteriophages, findings that can have a clinical impact in phage therapeutic approaches (treating bacterial infections with bacteriophages).