15 November 2021

CRISPR instead of bacteriophages

Plasmid with CRISPR/Cas9 killed pathogenic strains in mouse intestine

Anastasia Sverkunova, N+1

Scientists have created a plasmid with a high conjugation rate, which is transmitted to 96 percent of target bacteria in two hours and leads to the death of pathogenic strains in the mouse intestine. It has a built-in CRISPR/Cas9 complex that recognizes the target gene and cuts the chromosome of the cell. The new plasmid is transmitted twice as fast as the wild type that was used earlier. The study is published in Molecular Systems Biology (Neil et al., High-efficiency delivery of CRISPR-Cas9 by engineered probiotics enables precise microbiome editing).

Many enterobacteria, such as Escherichia coli, Klebsiella pneumoniae, Shigella sp. and Salmonella sp., can rapidly accumulate antibiotic resistance genes. Since some of them cause deadly infections, scientists are looking for alternative treatments that do not include antibiotics. One of these methods is the transportation of the CRISPR/Cas9 system into the target cell to cut the double—stranded DNA, which leads to the death of the bacterium. Bacteriophages or cells of other bacteria are used to deliver this system. Bacteriophages do deliver CRISPR/Cas9 quickly and accurately, but their selectivity is reduced by the rapid mutation of phage receptors on the cell surface, and environmental conditions in the intestine (in particular, acidity and enzyme activity) worsen their effectiveness. Gut-adapted bacteria are more resistant and use bacterial conjugation to deliver CRISPR/Cas9 to target cells, but previous experiments have shown low efficiency of plasmid transfer in the gut of mice.

To solve this problem, Canadian scientists from the University of Sherbrooke, led by Kevin Neil, have developed a strain of E. coli with a higher efficiency of bacterial conjugation. It contains a TP114 plasmid with a CRISPR/Cas9 system targeting the cat gene, thanks to which it is effective in treating mice from antibiotic-resistant strains of E.coli and Citrobacter rodentium. In a previous study, screening of most of the plasmids used showed that TP114 is most effectively transmitted to neighboring bacteria in the gut microbiota.

To begin with, the researchers created three strains of E. coli: KN01, KN02, KN03. All of them are resistant to streptomycin, but each has individual resistance to spectinomycin, chloramphenicol and tetracycline, respectively. Differences in resistance allowed these bacteria to be phenotyped during experiments. KN01 became plasmid transporters, KN02 became a target strain carrying this gene, and KN03 became a non—target strain (control).

To evaluate the effectiveness of conjugation, all three strains were injected into the intestines of female mice (C57BL line, six individuals in each group). 36 hours after the introduction of plasmid donor bacteria, the amount of the target strain decreased by 98.6 percent on average, similar results were obtained with the introduction of the donor strain 12 hours before the target. And ribotyping of all microorganisms indicated that after treatment with the KN01 strain, the number of ten highly conserved bacterial groups did not change.


Scheme of accelerated laboratory evolution.

To create more effective variants of the TP114 plasmid, the researchers used accelerated laboratory evolution, and the resulting mutant turned out to be twice as effective as the wild type. The authors of the work tried to transfer the same plasmid not to E. coli, but to Citrobacter rodentium. After two days, the number of pathogenic bacteria decreased by 96 percent.

Thus, the researchers showed that bacterial conjugation is a very accurate method of plasmid transmission: for example, in a constructed plasmid, the conjugation rate increased by three orders of magnitude compared to the wild type. And the high efficiency of destroying the target strain in the mouse intestine (up to 96 percent) indicates that the intestinal environment has little effect on conjugation. Moreover, the introduced bacteria did not affect the intestinal microbiota in any way, destroying only the target pathogen. The authors believe that targeting the system for antibiotic resistance genes may not be the best option for choosing a target gene, since they are not always found in chromosomal DNA. In the future, scientists suggest using conservative genes, for example, ribosomal proteins, to increase the probability of death of target cells.

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