CRISPR-Cas9 system that kills targeted pathogenic bacteria may be microbiome friendly.

By: Puck deRoos

Please join me in welcoming our new contributor Puck deRoos. She will be writing short articles about the latest microbiome papers. I am so excited to have you on the team! – Elisabeth Bik

New research has highlighted a potential system that can specifically target pathogenic bacteria without disturbing the rest of the microbial community1.  

Currently, the ways to treat pathogenic bacterial infections are non-specific and may have unintentional longer lasting effects because they often alter the composition of the gut microbiome. The rise of multi-drug resistant bacteria has shown the limitations of general broad-spectrum antibiotics2, while phage-based therapy has been associated with  a smaller but similar rise in phage resistant bacteria3,.  Stool transplants have shown promising results in treatment of particular intestinal conditions, but are not suitable for many other diseases. In addition, they completely alter the patients gut microbiome, which may have unknown long-term effects4

 

The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) Cas-9 (CRISPR ASsociated protein-9) system is able to target specific microbial species, and cause cell death due to double stranded breaks in the microbial DNA. In a recent paper published in Nature Communications, researchers have developed a method to specifically target microbial pathogens using a cis-conjugative CRISPR system where all the necessary machinery is encoded within one plasmid, or bacterial DNA sequence.

The cis-conjugative plasmid system has much higher rate of conjugation, which is how bacteria transfer genetic material between different cells, than its trans-conjugative counterpart, which has the CRISPR and conjugative sequences on different plasmids. One of the reasons the cis-conjugative system is more effective is that it is able to continue to work throughout multiple conjugations, so its effect increases exponentially throughout time. In contrast,  the trans system only works once. 

The rates of conjugation were measured from host bacteria Escherichia coli to bacterial recipient Salmonella enterica. The rate for the cis system continually increased and reached a maximum of 1 X 10-2 at 24hour, while the trans system maxed out early at ~1 X 10-3 but decreased to ~1 X 10-5 at 24hours. These results showed a ~1000-fold increase in the rates of the new cis-conjugative system compared to previous trans-conjugative system. 

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This new tool for targeting specific bacteria can be extremely useful within the human microbiome. Most microbial communities in our body exists as a biofilm, a community of different microorganisms that stick together on a surface. The new cis-conjugative system developed by researchers at Schulich School of Medicine and Dentistry has up to a potential 100% effective rate of conjugation with bacteria grown in a biofilm. The same cis system in a regular bacterial assay or filter based assay shows a ~500 to 1000-fold decrease in the rate of conjugation, increasing the systems usefulness in regards to the microbiome. 

In conclusion, this new method of specifically delivering a CRISPR-Cas-9 system to pathogenic bacteria without disrupting the composition of the entire microbial community is an exciting new direction for the targeted  treatment of microbial infections. 

 

1. Hamilton TA et al. Efficient inter-species conjugative transfer of a CRISPR nuclease for targeted bacterial killing. Nature Communications. 10, 4544 (2019). https://doi.org/10.1038/s41467-019-12448-3

2. Theuretzbacher, U. Antibiotic innovation for future public health needs. Clin. Microbiol. Infect. 23, 713–717 (2017). 

3. Chatain-Ly, M. H. The factors affecting effectiveness of treatment in phages therapy. Front. Microbiol. 5, 51 (2014). 

4. Khanna, S. et al. Changes in microbial ecology after fecal microbiota transplantation for recurrent C. difficile infection affected by underlying inflammatory bowel disease. Microbiome 5, 55 (2017).