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Slaying Superbugs With a New Type I CRISPR Gene-Editing Tool

A new type I-F CRISPR-Cas system has been developed to be transferrable to heterologous hosts and demonstrates promising efficiency, stability, and “scarless” gene-editing, opening up a new avenue to genetically edit various bacterial species and superbugs.

Researchers have successfully developed a transferrable and integrative type I CRISPR-based technology that can edit various clinical isolates of Pseudomonas aeruginosa, a superbug responsible for hospital-acquired infections. This new technique is expected to speed up the process of identifying the causative factors of multidrug resistance and accelerate the development of anti-resistant strategies.

Since the CRISPR-Cas system was first adapted from a naturally occurring genome editing system in bacteria, this gene-editing tool has taken the world of genetic engineering by storm and enabled numerous medical breakthroughs in a way only few biological innovations have before. The basis of this unique technology is the Class 2 type II CRISPR/Cas9 system, which can hone on and precisely cut a specific DNA sequence, thus allowing scientists to insert, delete, or modify genes, before finally letting natural DNA repair enzymes take over and re-join the spliced ends.

Despite being the most widely-used genome-editing tool today, the applications of the Class 2 CRISPR-Cas system to edit bacterial genomes are limited. The system represents only 10 per cent of all the CRISPR-Cas systems naturally encoded in prokaryotes, with many others that remain to be identified.

Making up for about 50 per cent of all CRISPR-Cas systems identified to date, the type I system is the most widely distributed. Its abundance, high specificity, minimal off-targeting, and ability to make large fragment deletions, all of which are distinctive advantages inaccessible with the Class 2 systems, experts consider type I as a deep reservoir for the expansion of CRISPR-based toolkits. However, type I CRISPR-Cas systems hinge on a multi-component effector complex, known as Cascade, to disrupt DNA. Unfortunately, Cascade is not readily transferrable to heterologous hosts, thus limiting its applications for genome editing and therapeutics.

In their previous research, Dr. Aixin Yan and colleagues from the University of Hong Kong identified a highly active type I-F CRISPR-Cas system in a clinical multidrug-resistant strain of P. aeruginosa, PA154197. Based on the native type I-F CRISPR Cas system, the researchers developed a genome-editing method that can rapidly identify the determinants of resistance in the multidrug-resistant clinical isolate P. Aeruginosa.

In the present study published in Nucleic Acids Research, the team found a way around the barrier of transferring the complex type I Cascade to heterologous hosts. They first cloned the entire type I-F Cas operon into an integration proficient vector, mini-CTX. The researchers then delivered the cassette into heterologous hosts via conjugation. The mini-CTX vector allowed for the integration of the entire Cascade onto the conserved attB genetic locus in the genome of the heterologous hosts. This enables the new hosts to harbour a functional, “native” type I-F CRISPR-Cas system that can be stably expressed.

The team took their experiments a step further and demonstrated the efficacy of the type I-F Cascade. Their reports revealed that the type I-F Cascade displayed a significantly greater DNA interference capacity and higher strain stability compared to the conventional transferrable Cas9 system. Their type I-F Cascade system also showed a remarkable 80 per cent efficiency when used for genome editing and is much simpler, requiring only a one-step transformation of a single editing plasmid. Furthermore, they were able to achieve scarless genome editing in host cells as the type I-F Cascade-mediated deletion of large DNA fragments can readily remove the introduced type I-F Cascade genes from the host genomes.

To improve the versatility of their technology, the scientists also upgraded the platform to create an advanced, transferrable system that includes a highly active type I-F Cascade and a recombinase to promote the application of the system in other strains with poor homologous recombination capacity, wild P. aeruginosa isolates that have not been sequenced, and in other Pseudomonas species.

By adding another valuable tool to the gene-editing kit, scientists hope to genetically edit other wild bacterial species and isolates that have clinical and environmental significance. Dr. Yan, for one, predicts that the system will be extended to editing not only pathogens but also the human microbiome to promote health, saying, “We believe that CRISPR-based technology and therapies will bring new hopes to combatting superbugs in the future.” [APBN]

Source: Xu et al. (2021). A transferrable and integrative type I-F Cascade for heterologous genome editing and transcription modulation. Nucleic Acids Research.