GenDels · Development of a new CRISPR-Cas3-based tool for large genomic deletions

Development of a new CRISPR-Cas3-based tool for large genomic deletions

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.

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