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CRISPR- Cas9: A Promising Tool against antibiotic-resistant bacterial infections
Antibiotic-resistant bacterial infections are a growing global crisis, causing millions of deaths worldwide. Bacteria develop resistance through two main mechanisms: random mutations in their DNA or acquiring resistance genes from other bacteria via horizontal gene transfer (HGT). Horizontal gene transfer is a process where genetic material (DNA or RNA) is exchanged between bacteria, either replacing existing genes or introducing new ones. Both mutations and gene transfer can enhance efflux pump production to expel antibiotics, enable enzymatic breakdown of antibiotics, or introduce mechanisms that block antibiotic activity. These adaptations allow bacteria to survive antibiotic exposure, making infections harder to treat. As antibiotic-resistant strains continue to rise, alternative approaches like probiotics are gaining attention.
Probiotics as an Alternative to Antibiotics
Probiotics are beneficial microorganisms that have shown promise in treating gastrointestinal conditions like irritable bowel syndrome and antibiotic-associated diarrhea. Researchers are also exploring genetically engineered probiotics for conditions such as cancer and inflammatory bowel disease. By using genetic engineering, probiotics can be modified to enhance their natural functions or introduce new therapeutic properties, offering health benefits beyond conventional probiotics.
E. coli Nissle 1917 (EcN), a well-established probiotic, has recently gained attention as a stable platform for developing living therapeutics, drug delivery systems, and industrial microbial applications. However, a major concern is that engineered probiotics may acquire antibiotic–resistant genes through horizontal gene transfer. Protecting probiotic strains and ensuring the containment of bioengineered probiotics is crucial.
A promising solution is the use of CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats – CRISPR-associated proteins) as a defense system against horizontal gene transfer. A recent study explored the use of CRISPR to enhance genetically engineered probiotics. By incorporating CRISPR into drug-resistant bacteria, the system can inactivate them or eliminate antimicrobial resistance, offering a potential strategy to control the spread of antibiotic resistance.
CRISPR-Cas: A Defense System Against Horizontal Gene Transfer
CRISPR-Cas9 is a bacterial immune system that protects against viral infections. It is a family of DNA sequences derived from past bacteriophage (virus) infections, allowing bacteria to recognize and destroy similar viruses upon reinfection. CRISPR-Cas plays a key role in defending bacteria from foreign genetic material, including bacteriophages and potentially harmful plasmids.
CRISPR-Cas has two main functions: detecting and cutting DNA. It consists of two key components:
- Guide RNA (gRNA): A small RNA sequence that directs the system to a specific DNA target.
- Cas protein: A specialized enzyme (like Cas9) that acts as molecular scissors, cutting the target DNA.
This system operates in three main stages:
- Adaptation (Acquisition)
When a bacterium encounters foreign DNA, Cas proteins cut small fragments of it and integrate them into the CRISPR array as spacers. These spacers serve as a genetic memory of past infections. - Expression (Processing)
The CRISPR array is transcribed into a long RNA molecule, which is then processed into smaller CRISPR RNAs (crRNAs). These crRNAs pair with another RNA molecule called tracrRNA, forming a complex with the Cas protein. - Interference (Targeting & Cleavage)
When the same virus reinfects the bacterium, the guide RNA directs the Cas protein (e.g., Cas9) to the matching viral DNA. The Cas protein then cuts the viral DNA, stopping the infection before it can spread.
This precise ability to target and cut specific DNA sequences makes CRISPR-Cas9 a powerful tool for genetic engineering, allowing scientists to edit genes in bacteria, plants, animals, and even humans.
How does CRISPR-Cas9 protect the bacteria from acquiring resistant genes?
The present study demonstrated the efficiency of CRISPR-Cas9 in limiting the acquisition of antibiotic-resistant genes. However, the transfer of genetic elements encoding virulence factors poses a risk, as non-pathogenic probiotic strains could become harmful. With ongoing advancements in bacteria-based drug delivery for conditions like inflammatory bowel disease, the demand for safer probiotics is expected to increase.
To prevent the spread of antibiotic resistance, a CRISPR-Cas9 system was designed to target resistance genes on a Klebsiella pneumoniae plasmid (a small circular DNA found in bacteria that can replicate independently) linked to a 2005 outbreak in Sweden. The system used 16 spacer sequences to recognize eight common antimicrobial-resistance genes in bacteria. The CRISPR array, tracrRNA, and cas9 were driven by strong synthetic promoters for effective gene targeting. A control without the targeting spacers was included. Built on a plasmid for easy use, the system was tested in E.coli and showed promise in reducing antimicrobial-resistant gene transfer.
The CRISPR-Cas9 system blocks bacteria from acquiring antibiotic-resistant genes. To test this, plasmids carrying one of eight targeted genes into E.coli cells were introduced. Four control genes that were not targeted by the CRISPR construct were also included. These genes were placed on a plasmid and transformed into E.coli strains carrying either an active CRISPR-Cas9 system (pCRISPR+) or a control version missing the targeting spacers (pCRISPR-).
The results showed that pCRISPR+ completely blocked the uptake of DNA with targeted resistance genes, while bacteria with pCRISPR- acquired 10,000 to 100,000 copies of the genes.CRISPR did not affect the control genes, proving that it specifically blocks antibiotic-resistance genes from spreading. In conclusion, our CRISPR-Cas9 system can serve as a valuable defense mechanism, alongside other strategies, to further reduce the risk of unwanted gene acquisition in genetically modified probiotic microorganisms and is a powerful tool against antibiotic-resistant bacterial infections.
Featured image: AI-Generated
Additional sources:
1. Li, T., Yang, Y., Qi, H., Cui, W., Zhang, L., Fu, X., He, X., Liu, M., Li, P., & Yu, T. (2023). CRISPR/Cas9 therapeutics: Progress and prospects. Signal Transduction and Targeted Therapy, 8(1), 1-23. https://doi.org/10.1038/s41392-023-01309-7.
2. Keeling, P. J., & Palmer, J. D. (2008). Horizontal gene transfer in eukaryotic evolution. Nature Reviews Genetics, 9(8), 605-618. https://doi.org/10.1038/nrg2386
3. Yu M, Hu S, Tang B, Yang H, Sun D. Engineering Escherichia coli Nissle 1917 as a microbial chassis for therapeutic and industrial applications. Biotechnol Adv. 2023 Oct;67:108202. doi: 10.1016/j.biotechadv.2023.108202. Epub 2023 Jun 19. PMID: 37343690.
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