New CRISPR Delivery Mechanism Allows Specific Bacteria to Be Targeted to Alter the Microbiome

New CRISPR Delivery Mechanism Allows Specific Bacteria to Be Targeted to Alter the Microbiome

A new method of delivering CRISPR has been developed that allows scientists to deliver the gene editing tool into microorganisms and potentially develop highly personalized treatments for a wide range of diseases and health conditions.

CRISPR – Clustered Regularly Interspaced Short Palindromic Repeats – is a system used by bacteria as a defense against genetic modifications by viruses. CRISPR is a family of DNA sequences that are derived from bacteriophages that have previously infected the bacteria. They are used to identify and destroy foreign DNA during subsequent infections. CRISPR is used to identify the foreign DNA and an associated enzyme – Cas9 for example – is used to cut the DNA and remove the foreign DNA.

By harnessing this mechanism, CRISPR can be used to search for specific genetic modifications or faulty genes and remove them from cells. Any disease caused by a faulty gene or mutation could potentially be corrected using this system. The problem, however, is how to deliver CRISPR into cells. Current techniques only allow CRISPR to be delivered to specific cells and it has not been possible to use CRISPR to target specific bacterial strains.

A new technique has now been developed by researchers at the University of Western Ontario that allows CRISPR to be delivered virtually anywhere, opening up a huge range of new applications. In addition to broadening the possibilities for gene editing using CRISPR, the researchers say their technique improves the efficiency of gene editing.

The new delivery mechanism could be used in next-generation non-antibiotic anti-microbial treatments for antibiotic-resistant bacteria such as Escherichia coli, Staphyloccous aureus, or Neisseria gonorrhoeae. The researchers also suggest CRISPR could be used to change the gut microbiome.

The new delivery mechanism harnesses the same method that bacteria use to replicate, which allowed the researchers to deliver CRISPR to specific bacteria – A process called bacterial conjugation. This process allows bacteria to transfer genetic material between bacterial cells that are in contact with each other, via a bridge-like connection between the cells called a pilus. Through the pilus, genetic material such as a plasmid or transposon is transferred.

The researchers used plasmids based on the IncP RK2 conjugative system as the delivery vectors for a TevSpCas9 dual nuclease. The researchers used cell cultures that enhanced cell-to-cell contact and achieved conjugation rates approaching 100%. In the lab, the researchers were able to use this delivery mechanism to achieve high killing efficiencies for E.Coli and S. enterica, two bacterial strains that can cause food poisoning in humans.

“We were able to show near complete transfer of the delivery vehicle to another bacterial species under conditions where they are in intimate contact — in a biofilm,” said Gregory Gloor, PhD, Professor at Schulich Medicine & Dentistry, and co-author of the study. “This is important because biofilms are the natural state of the majority of bacteria, and being able to transfer DNA under these conditions is typically difficult, but we found a way to make it easy and efficient.”

The study is detailed in the paper – Efficient inter-species conjugative transfer of a CRISPR nuclease for targeted bacterial killing – which was recently published in Nature Communications. DOI: 10.1038/s41467-019-12448-3

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