Using CRISPR/Cas to develop biosafety materials

By Dr. Liji Thomas, MDReviewed by Benedette Cuffari, M.Sc.Mar 21 2022

The diagnosis and treatment of viral infections, especially those that cause large-scale outbreaks, is urgently needed, as the lack of these measures allows epidemics to progress. A new Biosafety and Health study discusses the use of the highly efficient clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) technology to identify viral genome sequences in these areas.

Study: Biosafety Materials: Ushering in A New Era of Infectious Disease Diagnosis and Treatment with the CRISPR/Cas System. Image Credit: Immersion imagery / Shutterstock.com

Introduction

Biosafety materials are materials that can prevent and mitigate problems with biosafety, which is an umbrella term for security breaches linked to biological pathogens or events. The use of these materials can protect humans from invasion by a novel pathogen, while also mediating a rapid response by the host. Other applications, such as delivering therapeutic and diagnostic drugs and reagents, are also possible.

The ongoing pandemic of the coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), proved to be a significant challenge to public health and the global economy as a result of its rapid transmission and different routes of transmission. To counter this, it was essential for researchers to develop diagnostics that were efficient, suitable, and inexpensive, as well as easily adapted to new SARS-CoV-2 variants.

The delivery of CRISPR/Cas systems through viral and non-viral vectors has been associated with several issues due to mutations and immunogenicity of the vectors. Non-viral vectors, for example, are not suitable for handling the delivery of large genes with greater safety and in a more targeted fashion.

Areas of use

The limitations of standard diagnostic testing, especially the widely used reverse transcription-polymerase chain reaction (RT-PCR) assay, include the need for sophisticated laboratories with biosafety standards, highly trained personnel, and standardized specific reagents and equipment. These criteria can cause delays in the detection and classification of infections.

Comparatively, CRISPR/Cas enables portability and readability and, when combined with RT-PCR, could help provide timely detection if used with biosafety materials for point-of-care sampling and testing. The cleavage of the genetic sequence in a highly specific manner by Cas9 will allow genotyping as well as the identification of single-nucleotide polymorphisms (SNPs) at specific sites.

Previously, a prototype was created for the detection of the Zika virus using SNPs found only in the American genotype. A paper-based field-ready diagnostic kit was developed within a week and tested on the field, where it provided results within three hours of testing. This technology can be adapted to numerous pathogens, thus overcoming the lack of accuracy of lateral flow tests.

SHERLOCK

One CRISPR/Cas13-based nucleic acid detection system called specific high-sensitivity enzymatic reporter unlocked (SHERLOCK) is another CRISPR-based system that uses Cas13a to cleave ribonucleic acid (RNA) side chains. When used to detect nucleic acids, SHERLOCK can detect a wide array of infections, such as the four serotypes of dengue virus and regional strains of Zika virus. It is a sensitive and specific tool that can be used for rapid field diagnosis.

SHERLOCK has been since updated, with a 3.5-fold enhancement in its sensitivity due to the incorporation of another nuclease triggered by CRISPR that can enhance the Cas13a signal.

Other CRISPR-based systems

Cas12a is a nonspecific double-stranded DNA cleavage enzyme that has been used to detect nucleic acid within an hour at very low costs. This system is referred to as 1-h low-cost multipurpose highly efficient system (HOLMES). Another Cas12a-based technique is the DNA endonuclease-targeted CRISPR trans reporter (DETECTR) system that provides 100% detection of human papillomavirus (HPV)-16.

Several other types of biosafety materials are being developed or are in use. For example, lipid nanoparticles (LNPs) are being used to deliver a variety of nucleic acids due to their low toxicity and ability to cross the cell membrane. The use of lipids with specific properties allows the self-assembly of LNPs into nanoparticles when in an aqueous environment to then disassemble and release their cargo in acidic surroundings.

One type of LNP system is used to treat hepatitis due to hepatitis B virus (HBV) by inhibiting HBV DNA replication. Another system uses similar particles to inject Cas9 messenger RNA (mRNA)/subgenomic (sg) RNA into a complex to knock out a specific gene. Others have employed cationic lipids to target genes that regulate autosomal dominant hearing loss in the hair cells of the ear through Cas9 ribonucleoproteins (RNPs).

LNPs may also deliver mRNA and plasmids of certain bacteria to combat HBV infection. Viral and non-viral vectors can be combined to enhance the packaging capacity. In this way, LNPs and adeno-associated viruses (AAVs) were loaded with Cas9 mRNA and repair/sgRNA, respectively, to treat genetic liver disease,

CRISPR/Cas systems are also useful in treating antibiotic-resistant infections by editing essential genes in the bacteria, thus reducing their growth. The Cas9 protein is modified covalently with cationic polymers that subsequently form nanocomplexes with specific sgRNA that resist antibiotic action. Similar products have been shown to experimentally inhibit HPV growth, cervical cancer cell growth, and reverse oncogenic changes.

Peptides are another platform that can be used to deliver Cas9-sgRNA to prevent respiratory infection by allowing the complex to be taken up by the respiratory epithelium. The Endo-Porter (EP), an amphipathic helical peptide, is another example that has been used to enhance catabolism and adipocyte browning, thus releasing bioactive factors and promoting energy utilization to treat metabolic diseases.

Many other applications have been explored, including the gene-editing of stem and progenitor cells of the hemopoietic series in the bone marrow to treat certain diseases. Gold nanoparticles with Cas9 RNP, donor DNA, and an endosomal polymer have been used for homology-directed repair in embryonic stem cells, induced pluripotent stem cells, and mice showing symptoms of Duchenne muscular dystrophy.

Extracellular vesicles carrying various types of biomolecules are another important part of intercellular signaling. For example, the Gesicle from vesicular stomatitis virus G glycoprotein is designed to transport Cas9 RNPs into the cell to prevent viral replication by editing the viral genome.

Future directions

The potential utility of biosafety materials remains to be harnessed for reducing the transmission of infectious diseases, particularly in low-resource settings. This technology would be particularly useful in detecting pathogenic microbes and preventing future outbreaks from progressing to the scale of the current COVID-19 pandemic. Ensuring food hygiene by detecting the presence of microbial contamination is one such method of food safety.

The treatment of genetic and metabolic conditions is another promising application of this technology. Certainly, Cas9 RNPs are safer and more specific than plasmids in the delivery of therapeutic proteins, with no toxicity to normal host tissues or organs.

It is necessary to design biosafety materials using materials that not only have the ability to carry plasmid DNA, mRNA, and Cas RNPs but also do not produce immune responses.”

The prevention of off-target effects is a high-priority area that must be addressed to make the use of these systems practical. With proper and thoughtful design, the CRISPR-Cas gene-editing platform could allow precise gene manipulation for the diagnosis and treatment of many clinical conditions.