The Evolution of CRISPR Technology in Combatting Antimicrobial Resistance

Assistant Professor Ibrahim Bitar of the Department of Microbiology, Faculty of Medicine, University Hospital in Plzen, Charles University in Prague, Plzen, Czech Republic, provided an overview of the molecular biology of CRISPR technology and highlight its potential applications in combating antimicrobial resistance in the second new research review.

Numerous bacteria have extensive CRISPR-associated genes (cas) and clustered regularly interspaced short palindromic repeats (CRISPRs) in their genomes. These genes serve as a defense against external invaders like viruses and plasmids.  The small sequences that make up the CRISPR arrays are repeating arrays that precisely match and originate from a nucleic acid sequence that was previously invading the host.

Four to ten CRISPR-associated genes (cas), which encode the Cas proteins and are highly conserved, go hand in hand with CRISPR sequences. Cas proteins use immunological memories stored in the CRISPR array to conduct adaptive immunity in prokaryotes, or bacteria.

The CRISPR/Cas system recognizes and breaks down the same external DNA components during subsequent invasions. It does this by integrating a little piece of foreign DNA from invaders like plasmids and viruses into its own repeat sequences.

Genotyping can be used to determine the clonality and origin of the isolates and classify them as a population of strains that were exposed to the same environmental conditions, such as geographic location (region) and community/hospital settings, and eventually further extended to track pathogenic bacteria around human society because the CRISPR/Cas systems integrate DNA from invasive pathogens in chronicle order.

CRISPR/Cas systems can be utilized as well to create antimicrobial agents; the introduction of self-targeting crRNAs will efficiently and selectively destroy target bacterial populations. Due to a scarcity of effective antimicrobial agents for treating multidrug-resistant (MDR) infections, researchers began looking for alternate strategies to combat MDR infections rather than going through the lengthy process of generating new antimicrobial agents that can take decades.

As a result, the notion of CRISPR/Cas-based selective antimicrobials was developed and demonstrated in 2014. Vectors coding Cas9 and guide RNAs targeting genomic loci of a given bacterial strain/species can be transmitted to the target strain via bacteriophages or conjugative strains. In principle, the modified CRISPR/Cas systems should eradicate target strains from the bacterial community, however, this is not always the case.

While these systems appear to be targets for manipulation/intervention, all bacteria are controlled by several routes to ensure they maintain control over the process. As a result, employing this system as an antimicrobial agent continues to pose significant hurdles.

Most approaches rely on conjugation to deliver the re-sensitized system; the vector is carried by a non-virulent lab strain bacteria that is expected to spread the vector/plasmid via conjugation. The conjugation process is a natural mechanism in which bacteria share plasmids (even with different species). The proportion of conjugated (successfully delivered) bacteria in the entire bacterial population is crucial for re-sensitized efficiency. This process is regulated by multiple complex routes.

Additionally, bacteria have internal anti-CRISPR systems that can fix any harm that CRISPR-Cas systems might have caused. Experts speculate that mobile genetic elements (MGEs) organize their counterdefense strategies in “anti-defense” islands. For example, acr, a gene that functions as a repressor of plasmid conjugative systems along with other similar variants, frequently clusters with antagonists of other host defense functions, such as anti-restriction modification systems.

Defense systems employed by the bacteria to protect itself from foreign DNA frequently co-localize within defense islands (genomic segments that contain genes with similar functions in protecting the host from invaders) in bacterial genomes.

In summary, this method seems very promising as an alternative way of fighting antimicrobial resistance. The method uses the concept of re-sensitizing the bacteria in order to make use of already available antibiotics – in other words, removing their resistance and making them vulnerable again to first-line antibiotics.

Ibrahim Bitar, Assistant Professor, Department of Microbiology, Charles University in Prague

He concluded, “Nevertheless, the bacterial pathways are always complicated and such systems are always heavily regulated by multiple pathways. These regulated pathways must be studied in depth in order to avoid selective pressure favoring anti-CRISPR systems activation, hence prevalence of resistance in a more aggressive manner.”