How Is CRISPR Gene Editing Being Used in Infectious Disease Research?
Alongside its cell and gene therapy applications, CRISPR technology is becoming an increasingly popular tool for infectious disease research.
Complete the form below to unlock access to ALL audio articles.
CRISPR gene editing has enabled scientists to rewrite the genetic code of living organisms and is revolutionizing medicine. In 2023, Casgevy became the first US Food and Drug Administration (FDA)-approved therapy that utilizes CRISPR-Cas9, offering new hope for patients with sickle cell disease.
Alongside its cell and gene therapy applications, CRISPR technology is becoming an increasingly popular tool for infectious disease research. It has allowed scientists to improve their understanding of the biology and genetics of human pathogens, and is being explored as a technique for diagnosing and treating diseases such as human immunodeficiency virus (HIV). Here, we highlight some of the latest applications of CRISPR gene editing in infectious disease research.
Could CRISPR gene editing provide a cure for HIV?
Retroviruses like HIV cleverly integrate their genetic material into host genomes and are notoriously difficult to treat. Even with effective treatment, some immune cells go into a resting state but still contain HIV DNA.
Infection with HIV is currently treatable with lifelong antiviral therapy to reduce viral load to undetectable levels, but it is not curable. CRISPR has provided new hope in the search for a HIV cure, and researchers are working towards using this technology to completely excise the viral DNA from the genome of host cells.
Scientists at Temple University published evidence last year showing that a single injection of a novel CRISPR gene-editing treatment safely and efficiently removes simian immunodeficiency virus (SIV) from the genomes of rhesus macaque monkeys.
The outcomes of this study set the stage for an ongoing Phase 1/2 clinical trial of EBT-101, a HIV-specific CRISPR-Cas9 gene-editing therapy, which was granted FDA Fast Track Designation in July 2023. The preclinical study, published in the journal Gene Therapy, represented a significant advance in the generation of a cure for HIV in humans.
What is EBT-101?
EBT-101 is an in vivo CRISPR-based therapeutic candidate designed to excise HIV pro-viral DNA from HIV-infected cells. The treatment employs CRISPR-Cas9 and two guide RNAs that target three sites within the HIV genome, thereby excising large portions of the HIV genome.
In a proof-of-concept study (the results of which are yet to be peer reviewed) presented at the 2024 European Congress of Clinical Microbiology and Infectious Diseases (ECCMID), researchers claimed they removed HIV from lab-cultured cells using CRISPR-Cas9 gene editing.*
According to the scientists, they adopted a broad-spectrum approach, using CRISPR technology to edit two regions of the HIV genome that are conserved across all known strains of the virus. They found that the size of the vector used to transport the cassette encoding the therapeutic CRISPR-Cas reagents into the cells was too large. Another challenge was reaching the HIV reservoir cells that rebound when HIV antiretroviral treatment is stopped.
To overcome these challenges, the authors tried various techniques to reduce the size of the cassette and therefore the vector system itself. They successfully minimized the size of the vector, enhancing its delivery to HIV-infected cells, and were able to target HIV reservoir cells by focusing on specific proteins found on the surfaces of these cells.
The authors stated, “We have developed an efficient combinatorial CRISPR attack on the HIV virus in various cells and the locations where it can be hidden in reservoirs, and demonstrated that therapeutics can be specifically delivered to the cells of interest.”
The researchers hope to advance to preclinical models to study the safety and efficacy of a therapeutic strategy combining CRISPR therapeutics and receptor-targeting reagents.
CRISPR enzymes support proactive planning against future pandemics
Searching for ways to improve CRISPR-based solutions to RNA viruses, which could help combat future pandemics, is an active area of research.
CRISPR-Cas13 systems have become indispensable tools for various RNA targeting applications, including antiviral development to combat viruses such as SARS-CoV-2.
Examples of Cas enzymes
Many CRISPR associated proteins (Cas) possess nuclease activity and play a vital part in the bacterial and archaeal defense system. In 2005, the first Cas protein with nuclease activity was discovered while studying the genome of Streptococcus thermophilus. The protein is now known as Cas9 and is one of the most common Cas proteins used in CRISPR gene-editing of DNA. Other popular systems include the CRISPR-Cas13 system, which is used for precise RNA manipulation without permanent changes to the genome. Cas12 and Cas14 enzymes are also being explored in genome editing technologies.
Within the Cas13 family, Cas13d is the most active subtype in mammalian cells. However, it is inefficient in the cytoplasm of cells, where many RNA viruses replicate.
Researchers from Helmholtz Munich and the Technical University of Munich overcame this obstacle by engineering nucleocytoplasmic shuttling Cas13d (Cas13d-NCS). This system can transfer nuclear CRISPR RNA into the cytosol.
The scientists showed that Cas13d-NCS outperforms its predecessors in degrading mRNA targets and neutralizing self-replicating RNA, including replicating sequences of several variants of SARS-CoV-2.
This achievement represents a significant step in strengthening our defenses against future outbreaks of RNA viruses.
Disease vectors: CRISPR addresses the root of the problem
Many human pathogens such as malaria are vector-borne. Vectors are living organisms that can transmit infectious pathogens between humans. Vector-borne diseases are human illnesses caused by parasites, viruses and bacteria that are transmitted by vectors. These diseases are often transmitted from blood-feeding arthropods like mosquitoes. CRISPR gene editing provides an opportunity to control the spread of these animal vectors, thus preventing the transmission of the pathogens they carry.
Chagas disease can be transferred to humans by insects such as triatomine bugs, also known as kissing bugs. As treatment options are limited, strategies for Chagas disease control have focused on ways to manipulate the organisms that carry the parasite.
The application of CRISPR technology in kissing bugs has proven difficult. Traditional gene editing methods involve injecting the CRISPR gene-editing material directly into embryos, which, due to the hardness of kissing bugs' eggs, has proven challenging.
In a recent paper published in The CRISPR Journal, researchers demonstrated the application of CRISPR-Cas9 gene editing in kissing bugs for the first time, creating new possibilities for using genetic technologies to control vector-borne Chagas disease.
The research team from Penn State College of Agricultural Sciences have developed an approach called Receptor-Mediated Ovary Transduction of Cargo or “ReMOT Control”. This technology enables the injection of materials directly into the mother's circulatory system and guides that material to the developing eggs.
“Here, we showed that you could genetically modify this vector insect. Our technology has the potential to make gene editing more efficient, easier and cheaper in a wide range of animals,” said Dr. Jason Rasgon, Dorothy Foehr Huck and J. Lloyd Huck Endowed Chair in Disease Epidemiology and Biotechnology at Penn State College of Agricultural Sciences.
CRISPR improves the time to result in disease diagnosis
In addition to treating and preventing infectious diseases, CRISPR technology has been widely used in research developing novel diagnostic tools for diseases such as SARS-CoV-2.
A rapid test for diagnosing melioidosis, a rare tropical disease, was recently described in a study published in The Lancet Microbe.
Melioidosis is caused by the bacterium Burkholderia pseudomallei. Present in soil and water in tropical and subtropical regions, the bacterium enters humans via inoculation through skin abrasions, ingestion or inhalation.
“Melioidosis has been neglected despite its high mortality rate and high incidence in many parts of Asia. Early diagnosis is essential so that the specific treatment required can be started as soon as possible,” said Professor Nick Day, senior author and director of the Mahidol-Oxford Tropical Medicine Research Unit .
Diagnosis of melioidosis requires culturing bacterial samples, which takes three to four days. In this study, the team set out to develop a new rapid test to reduce patient diagnosis time.
Their test, called CRISPR-BP34, involves rupturing bacterial cells and using a recombinase polymerase amplification reaction to amplify the bacterial target DNA. Additionally, a CRISPR reaction is used to provide specificity, and a simple lateral flow read-out is employed to confirm cases of melioidosis.
The team collected clinical samples from 114 patients with melioidosis and 216 patients without the disease at a hospital in northeast Thailand, where the disease is endemic. The CRISPR-BP34 test was then applied to these samples.
The new test showed enhanced sensitivity at 93%, compared to 66.7% in the current gold-standard culture-based method. It also delivered results in less than four hours for urine, pus and sputum samples, and within one day for blood samples.
Eliminating antimicrobial-resistant bacteria with gene editing
Widespread misuse and overuse of antimicrobials have led to antimicrobial resistance (AMR) being declared one of the top global public health and development threats. Scientists across the globe are now rapidly searching for viable alternatives to antibiotics.
CRISPR has not only been used to identify AMR genes but has potential as a therapeutic tool to treat antibiotic-resistant bacteria and other pathogens.
At North Carolina State University, researchers showed that the CRISPR-Cas system can target and eliminate the gut bacteria Clostridioides difficile (C. difficile) in vivo. The results were published in the journal mBio.
Antibiotic use is a major risk factor for C. difficile infection because broad-spectrum antimicrobials disrupt the indigenous gut microbiota, decreasing colonization resistance against C. difficile.
The study showed that the CRISPR-Cas system in C. difficile can be repurposed as an antimicrobial agent through the expression of a self-targeting CRISPR that redirects endogenous CRISPR-Cas3 activity against the bacterial chromosome.
The researchers tested this approach in mice infected with C. difficile. Two days after the CRISPR treatment, the mice showed reduced C. difficile levels, however, those levels began to increase again two days later.
The researchers explained that future work will include retooling the phage to prevent C. difficile from returning after the initial effective killing.
The future of CRISPR in infectious disease research
The versatility of CRISPR gene editing has resulted in its application in many facets of infectious disease research. As CRISPR technology continues to undergo technical improvements, the prospects for its application in treating incurable diseases such as HIV are becoming increasingly promising. The notable applications discussed here merely offer a glimpse into the evolving landscape of how CRISPR gene editing can be harnessed to improve human health.
* These research findings are yet to be peer-reviewed. Results are therefore regarded as preliminary and should be interpreted as such. Find out about the role of the peer review process in research here. For further information, please contact the cited source.
