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CRISPR Antimicrobials: A bacterial defense strategy turned on itself

Phage introduction of CRISPR-Cas9, DNA break and eventual cell killing.

Phage introduction of CRISPR-Cas9, DNA break and eventual cell killing.

Bacteria are innovators. Life as a bacterium means lethal and frequent chemical assaults from antibiotic exposure and physical assault from the most abundant “biological entities” living on earth, bacteriophages. The myriad of resistance mechanisms used in response to these threats illustrates the creativity of bacterial survival. The CRISPR-Cas system is one shining example of how microbes resist killing by viruses. Bacteria recognize foreign DNA injected from viruses and degrade it, along with keep a record of all viral DNA sequences from previous infections, as a kind of ‘memory’ for the bacterial immune system.

 Components of a CRISPR system.

Encoded on the bacterial chromosome of numerous species, the CRISPR immune system has 3 main components:

  • An enzyme that cuts double stranded DNA (Cas gene)
  • A short RNA guide (tracRNA)
  • CRISPR array of “clustered regularly interspaced short palindromic repeat” sequences separated with “spacer” regions that contain the target DNA

The original function of CRISPR/Cas system is to degrade incoming plasmid or viral DNA, but it has been developed as a powerful genome engineering tool. It is easy and cheap to employ this pioneering system towards cutting any desired target DNA, which explains its widespread and rapid usage in many academic research labs. The CRISPR technology permits DNA cutting, editing, introducing mutations, deletions or insertions, with unprecedented precision. There can be “off-target”mutations that arise and this is an area where the technology needs refining to limit the creation of potentially harmful DNA changes.

Game changing applications of CRISPR and the patent race to own it.

The list of potential applications is growing and underscores its transformative potential. CRISPR may give the field of gene therapy a boost, with the ability to correct numerous human genetic diseases including blindness, Duchenne muscular dystrophy, Cystic Fibrosis, sickle cell anemia and others. CRISPR gene drives are being developed to engineer sterile mosquitoes to help eliminate vector-borne diseases like malaria, dengue fever, West Nile and Zika. Plants could be edited for resistance to drought and pathogens, which can proceed without the addition of any foreign genes, but rather editing existing genes. This approach to not introduce foreign genes offers a solution to the problems associated with GM organisms. The other huge opportunity is to engineer T cells to combat HIV infection or to treat cancer. Since HIV infects T cells, it is proposed to engineer T cells to either resist HIV infection, or to redirect HIV immune responses. As part of the oversimplified “cancer moonshot”, CRISPR is also being considered for use in treating cancer.

Despite the many hurdles faced to obtain these diverse goals, it is no surprise that there is a mad rush to patent the CRISPR technology and researchers at the Broad Institute (MIT) (Feng Zhang) and the University of California Berkeley (Dr Jennifer Doudna – along with Emmanuelle Charpentier) are engaged in a combative patent dispute. The value of the technology is difficult to estimate but the impact will be comparable to the invention of PCR by Kary Mullis, and will also likely lead to a Nobel prize. Professor, open science advocate, and co-founder of the Public Library of Science (PLOS) journals, Michael Eiesen, has been refreshingly honest and critical of the tactics used in the CRISPR patent debate. He even went so far as to propose that neither group should be able to patent a technology that was government funded research, and therefore ultimately owned by the tax-paying citizens.

Novel CRISPR antimicrobial treatment that does not involve antibiotics.

Widespread antibiotic resistance is an increasing, global problem due to the excessive use of antibiotics in hospitals and more importantly, in agriculture. The arms race between microbes and antibiotics is a losing battle for antibiotic discoverers. As soon as a new antibiotic is discovered and employed, there is often the immediate discovery of clinical resistant isolates in response to treatment. It has also been shown than antibiotic resistance even precedes the clinical usage of antibiotics, as bacteria need to defend against antimicrobial attack our in the real world. Resistance is out there.

As an alternative to continuing our search for new antibiotics (which is ongoing in new and exciting ways), scientists are developing new approaches to kill microbes and treat bacterial infections. The CRISPR-based antimicrobial approach described here is one such alternative approach to killing microbes without antibiotics. A bacterial defense strategy against viruses is being turned on itself.

Early work showed that the CRISPR-cas system can be directed to cleave DNA sequences in the bacterial genome, which led to irreparable damage and bacterial death (1). Recent studies have moved this concept further by developing a system to deliver cytotoxic, CRISPR-Cas9 systems to microbes. Phagemids are plasmids derived from phage genomes, viruses that specifically target bacteria. The phagemids have been engineered to express all the components of a CRISPR system, in addition to the information required to package the phagemid DNA into a phage particle. Bacteriophages are experts at landing on the outer bacterial surface, injecting its DNA into the bacterium, taking over the bacterial machinery and producing hundreds of viruses. The factory ultimately bursts, leading to death and release of an army of viruses to maintain the attack on neighbouring cells.

CRISPR Antimicrobials.

In back-to-back papers in Nature Biotechnology, two groups have published remarkably similar reports on the use of the Streptococcus pyogenes type II Cas 9 protein as a killing enzyme (2,3). The basic concept was to develop a CRISPR-mediated approach that can cause specific bacterial death. The element of specificity is particularly interesting in light of our increasing awareness of the dangers of broad-spectrum killing of healthy and helpful commensal, microbiome members. Drugs that cause little collateral damage is a new principal of antimicrobial design, which is a large shift from conventional thinking that broad-spectrum is best. Also, these papers explored the use of CRISPR to limit the transfer of antibiotic resistance genes.

Both approaches used phage-derived vectors to encode the CRISPR components of the the Cas9 enzyme, the short RNA guide (tracRNA) and a CRISPR array containing the desired cut sites within the spacer regions. When directing cleavage of kanamycin or ampicillin resistance genes, both groups could successfully show a loss of antibiotic resistance in the target organism, after delivery of the phagemid-encoded CRISPR systems. The same principal was applied to monitoring the efficiency of transformation, by measuring the number of viable cells in strains containing the target genes compared to strains that lacked the target. There was a 1000-fold decrease viability (and in the efficiency of conjugation) in hosts that contained the targeted cleavage site, indicating that successful DNA disruption and killing. Interestingly, there was never a complete killing of the target population in these experimental conditions.

To further examine the feasibility of this approach, various infection models were used to determine the capacity of CRISPR killing when applied to pathogens during an infection. In both a skin infection and the Galleria larval infection models, the pathogen numbers could be reduced by CRISPR-Cas antimicrobials, which permitted skin healing and survival of the larvae. The final experiments were to test the system when exposed to simple, mixed communities, in order to show specific pathogen targeting and non-targeting of commensals.

Although phages are being revisited for use as an alternative to antibiotics, engineered phages with CRISPR capacity have perhaps the added value of narrow spectrum killing. The biggest obstacle to improving these transmissable, programmed antimicrobials is to improve the method of delivery. It is not unreasonable to image a scenario where due to the low costs of DNA synthesis, custom, specific CRISPR-Cas-phage particles could be synthesized within a few days, and used as needed to treat any possible infectious disease.

References

1 . CRISPR design for next-generation antimicrobials. Beisel CL, Gomaa AA, Barrangou R. Genome Biol. 2014 Nov 8;15(11):516. doi: 10.1186/s13059-014-0516-x.

2.  Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials. Bikard D, Euler CW, Jiang W, Nussenzweig PM, Goldberg GW, Duportet X, Fischetti VA, Marraffini LA.                  Nat Biotechnol. 2014 Nov;32(11):1146-50. doi: 10.1038/nbt.3043..

3. Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases.Citorik RJ, Mimee M, Lu TK. Nat Biotechnol. 2014 Nov;32(11):1141-5. doi: 10.1038/nbt.3011.