CRISPR-Cas Gene Editing: A New Weapon against Infectious Disease

Infectious disease is a common cause of death worldwide, but the rise of antibiotic-resistant bacterial strains and lack of effective antiviral treatments means a potential future risk of increased mortality and global economic burden due to untreatable infections. This article discusses how CRISPR-Cas gene-editing technology is helping in the fight against increasingly resistant bacterial infections and rapidly mutating viruses—from facilitating a better understand of host-pathogen interactions and improving diagnosis, to potentially providing a new way to treat infectious disease.

The CRISPR revolution

The CRISPR locus (short for clustered regularly interspersed short palindromic repeats) was first discovered in E.coli in 1987 and found to be the basis of a bacterial adaptive immune system, providing prokaryotes with protection against foreign genetic material.¹ The CRISPR-Cas system has since been repurposed into a powerful but relatively simple programmable gene-editing technology. A short single-guide RNA molecule (sgRNA) guides the Cas9 endonuclease to the target site, where a double-strand break is introduced. This activates intrinsic cellular DNA repair pathways, either the non-homologous end joining (NHEJ) pathway that results in a disabling deletion of the target gene, or homologous repair (HR) that allows the integration of a donor sequence at the target locus. As well as gene knockout and targeted changes to the genetic sequence, gene expression can be regulated. Other modifications at the target site are also possible with the use of a catalytically inactive version of Cas9 (dCas9)—the expanded CRISPR toolkit also includes modulation of gene expression (CRISPRi and CRISPRa) and base editing.

Since the first CRISPR gene-editing experiments were demonstrated in 2012, the CRISPR-Cas9 technology has exploded into the biological sciences and been rapidly adopted by the scientific community. CRISPR has already shown promise in the prevention of malaria, tuberculosis, and herpes simplex virus.² Below, we highlight a few ways in which CRISPR has been recently applied to improving our understanding, treatment, and ongoing diagnosis of infectious disease.

Functional genomics with CRISPR to determine new antimicrobial targets

There is currently a distinct lack of new antibiotics and antiviral drugs making it to the clinic. Understanding host-pathogen mechanisms that govern how microbes induce pathogenesis is crucial for identifying new targets for rapid drug discovery and vaccine development. Soon after its debut, CRISPR-Cas9 was applied to functional genomic screening. Using a pooled sgRNA library workflow, CRISPR-Cas9 was successfully used at scale to enable high-throughput, genome-wide loss of function studies. CRISPR-Cas9 genome-wide screening has since been employed in a variety of pathogens to determine the molecular pathways that drive pathogenesis. These include identifying how the α-hemolysin virulence factor S.aureus causes cytotoxicity and genes involved in host-cell dependencies from Zika virus.^3,4

CRISPR-Cas9 as a next-generation diagnostic for antimicrobial-resistance genes

Since the discovery of penicillin in 1928, antibiotics have been the main treatment against bacterial infections, reducing mortality and significantly improving life expectancy the world over. But the ability of microbes to rapidly mutate and share genetic information, as well as the overprescription of antibiotics, has led to the emergence of superbugs—strains that are resistant to existing treatments. According to the UN, antibiotic resistance is thought to cause around 700,000 deaths per year, which could rise to 10 million by 2050.

Determining whether genes responsible for antimicrobial resistance (AMR) are present is crucial when formulating an optimal treatment strategy to limit the spread of drug resistance. Unfortunately, real-time metagenomic analysis is hampered by the low abundance of resistant pathogens against a high background. Recent work from the Crawford lab at the University of California, San Francisco used CRISPR to develop a novel NGS-targeted enrichment system called FLASH (finding low abundance sequences by hybridization), which they use as a diagnostic.⁵ By using sgRNA to guide Cas9 to AMR genes, those sequences are cleaved ready for next-generation sequencing. FLASH enables amplification of AMR targets and high levels of multiplexing and was shown to successfully identify AMR genes in patient samples, including those infected with pneumonia-causing bacteria and Plasmodium falciparum, the malaria parasite.

Selectively controlling the microbiome

The human microbiome is a complex ecosystem of species that all play a role in health —but treatment with broad-spectrum antibiotics to destroy pathogens also kills the “good” bacteria, upsetting the delicate balance and the positive symbiotic relationships that help control pathogens. This blunt instrument also provides a selective pressure that can lead to the further development of antibiotic-resistant strains.

In a recent Nature Communications paper, Hamilton et al used CRISPR-Cas9 to selectively target and kill Salmonella enterica but leave other bacteria species in a co-culture unharmed.⁶ Their work utilizes a conjugative plasmid to put the delivery machinery together with the necessary Cas9 molecules in a cis-conjugative system to selectively target essential genes in S. enterica cells and destroy them. The authors also suggest their new delivery system could be beneficial in controlling microbial imbalances on biofilms with potential applications in medicine, healthcare, and industrial processes—for example in treating Clostridium difficile, a hospital-acquired infection that is placing an increasing economic burden on healthcare systems worldwide.

CRISPR: as nature intended?

In nature, CRISPR is an endogenous bacterial system used to protect from foreign genetic material, so it makes sense that it is now being used in medicine against the bacteria themselves to fight infectious disease. And not only bacteria—recent work from the Broad Institute used the Cas13 protein from the CRISPR system to selectively target and destroy single-stranded RNA viruses, including Influenza A, significantly and rapidly reducing viral load and infectiousness.⁷

The programmable nature of the CRISPR gene-editing system means that as microbes continue to evolve and mutate, the CRISPR machinery can be quickly altered to destroy the new target as an antimicrobial drug, or detect it as a diagnostic. Work will now move onto demonstrate that these CRISPR-based antimicrobial applications can work in the clinic and help combat the growing specter of antimicrobial resistance and infectious disease.

Key Takeaways

• CRISPR is a gene-editing tool that has been repurposed from an endogenous bacterial immune system that protects from foreign genetic material
• Scientists can now use CRISPR to perform targeted and precise gene editing, with applications ranging from the improvement of agricultural crops, whole genome screening for drug discovery, as well as genome engineering for the treatment of human inherited disease
• CRISPR has also been utilized to discover new antimicrobial drug targets, understand the cellular mechanisms that cause disease, and identify whether antimicrobial resistance genes are present in a patient sample
• The revolutionary gene-editing technology may also provide a means to administer targeted therapy for specific bacterial and viral infections that can overcome the growing issue of antimicrobial resistance

References

1. Doudna, J. A. & Charpentier, E. The new frontier of genome engineering with CRISPR-Cas9. Science 346, (2014).

2. Doerflinger, M., Forsyth, W., Ebert, G., Pellegrini, M. & Herold, M. J. CRISPR/Cas9—The ultimate weapon to battle infectious diseases? Cellular Microbiology 19, (2017).

3. Winter, S. V., Zychlinsky, A. & Bardoel, B. W. Genome-wide CRISPR screen reveals novel host factors required for Staphylococcus aureus α-hemolysin-mediated toxicity. Scientific Reports 6,(2016).

4. Savidis, G. Identification of Zika Virus and Dengue Virus Dependency Factors using Functional Genomics. Cell Rep 16(1), (2016).

5. Quan, J. FLASH: a next-generation CRISPR diagnostic for multiplexed detection of antimicrobial resistance sequences. Nucleic Acids Res. 47(14), 2019.

6. Hamilton, T. A. et al. Efficient inter-species conjugative transfer of a CRISPR nuclease for targeted bacterial killing. Nature Communications 10, (2019).

7. Freije, C. A. et al. Programmable Inhibition and Detection of RNA Viruses Using Cas13. Molecular Cell (2019).