Date: 15th June 2020
At least 700,000 people die each year due to drug-resistant diseases and it is estimated by 2050 that such diseases could cause 10 million deaths per year. International organisations are therefore united in a demand for urgent action against such a devastating crisis and are calling for investment into research and development for new technologies to combat antimicrobial resistance. Now scientists report the development of a series of CRISPR-Cas13a-based antibacterial nucleocapsids for use as therapeutic agents against antimicrobial-resistant bacteria.
CRISPR-Cas systems are ushering in a new era of genome editing, and they becoming one of the most powerful tools in the arsenal of weapons fighting infection and diseases. Whilst Cas13 family members might be relatively new kids on the block, their roles as antivirals are starting to attract attention, in part because they cleave single stranded RNA (ssRNA). For example, we have recently seen the development of a CRISPR-Cas13-based approach, PAC-MAN (Prophylactic Antiviral CRISPR in huMAN cells), as a potentially pan-coronavirus antiviral which is able to inhibit SARS-CoV-2, and also CARVER (Cas-13-assisted restriction of viral expression and readout); a platform developed to detect and destroy single-stranded RNA viruses in human cells.
Now, here a team from the Jichi Medical University, the National Institute of Infectious Diseases, Japan, and the University of Glasgow, UK, have packaged programmed CRISPR-Cas13a into bacteriophage capsids to target antimicrobial resistance genes present in bacteria, creating a potent antibacterial agent.
So why Cas13a?
Whilst Cas13a has programmable RNase activity – via guide RNAs – once activated at it’s specific target site it also exhibits activity that leads to the non-specific degradation of any nearby RNA transcripts. This restricts host bacteria growth and it is thought to be a phage defence mechanism from the bacteria as it can limit the spread of the infection.
To begin the team lead by Longzhu Cui, established the bactericidal activity of Cas13a. Escherichia coli (E.coli) carrying the carbapenem resistance gene were transformed with a plasmid encoding Cas13a and crRNA spacer sequence (which forms the specific guide RNA) which targeted the resistance gene. A decrease in the number of bacteria by two to three orders of magnitude against the control bacteria was observed.
Packaging of CRISPR-Cas13a into bacteriophage capsid
In order to generate antibacterial agents the team packaged the CRISPR-Cas13a genes into bacteriophage. They created a series of CRISPR-Cas13a-based antibacterial nucleocapsids, termed CapsidCas13a(s).
Building on the initial experiment, the first series of CapsidCas13as were created to target Gram-negative bacteria, E.coli, and were designed to target four different genotypes of carbapenem-resistant genes and two colistin-resistant genes; all of which create antibiotic resistant problems in the clinic. The CapsidCas13as showed sequence specific bactericidal activity, whilst non-targeted controls did not, therefore the team concluded that these data indicated robust specificity for gene-directed antimicrobial therapy.
However, could the therapy be used to selectively kill target bacteria in a mixed population of antibiotic resistant bacteria? The answer here was yes, by preparing a mixed cell population containing control E.coli with two different resistant versions of E.coli, the team were able to show that each gene-targeting CapsidCas13a reduced the corresponding target cell population.
Whilst the system worked well as a Gram-negative antibacterial agent, the scientists also wanted to test whether it could function as a Gram-positive antibacterial too. Much in the same way, the team packaged the CRISPR-Cas13a constructs into a phage capsid – now using spacer sequences to target a resistance gene in Staphylococcus aureus (S.aureus). As one of the most prevalent antimicrobial resistant pathogens worldwide is methicillin-resistant S. aureus (MRSA) the team wanted to target the methicillin-resistant gene mecA. The system exhibited efficient mecA-specific bactericidal activity against MRSA, but not S. aureus strains deficient in mecA.
Conclusions and future applications:
The authors here have generated a new type of sequence-specific bacterial antimicrobial by leveraging the RNA cleavage activities of CRISPR-Cas13a. By delivering the system via carrier phage capsids, which are devoid of phage genome, it lends itself well to the translation into the clinic.
However, it should also be noted that in addition to its antimicrobial properties the team also adapted the system to detect specific bacteria. One emerging problem in the clinical is the increasing presence of ‘stealth’ resistant bacteria which are not identifiable by existing antibiotic susceptibility tests. The strength here, unlike other Cas-based systems currently available, is that this system does not require nucleic acid amplification and optic devices. However, limitations do still exist, such time-to-result which may be slow due to bacterial culture growing times.
Whilst, other Cas proteins have been explored as antibacterial agents, in the main they do not kill the bacteria. As many of the resistant genes are carried on plasmid DNA, DVA cleavage of these plasmids does not result in bacterial death. Here, the promiscuity of Cas13a once activated is the key to its success and the demise of the bacteria.
Whilst these are early days for the tech, there are many questions surrounding its safety and practical applications still to be answered. However, this new application has tremendous potential to solve some of our most pressing clinical dilemmas. Indeed, the work here yet again expands the practical applications that fall under the CRISPR-Cas umbrella, with the more well-established relative Cas9 currently under clinical trials scrutiny for gene editing purposes; their use in the diagnostic field rapidly increasing; and the use of CRISPR-Cas12 and 13 as antivirals, we can now add Cas13 as a highly specific antibacterial agent. Let’s hope this tech can help in the fight against some of the most deadliest antimicrobial-resistant bacteria.
Kiga, K., X.-E. Tan, R. Ibarra-Chávez, S. Watanabe, Y. Aiba, Y. Sato’o, F.-Y. Li, T. Sasahara, B. Cui, M. Kawauchi, T. Boonsiri, K. Thitiananpakorn, Y. Taki, A. H. Azam, M. Suzuki, J. R. Penadés and L. Cui (2020). “Development of CRISPR-Cas13a-based antimicrobials capable of sequence-specific killing of target bacteria.” Nature Communications 11(1): 2934.
https://doi.org/10.1038/s41467-020-16731-6
