A few years ago, the diagnosis of transthyretin amyloidosis, a slowly progressing disease that involves the buildup of abnormal protein deposits in the body’s organs and tissues was a death sentence for the patient.

Three decades of CRISPR research has changed that and revolutionized the treatment of “Untreatable Genetic Diseases.” Treating genetic diseases is no longer science fiction as in the 1900’s, thanks to CRISPR-Cas9 gene editing technology. CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats  and works in conjunction with Cas proteins (CRISPR associated proteins) to confer antivirus immunity in prokaryotes like bacteria and cyanobacteria by cleaving the viral genetic material.

The CRISPR-Cas9 system is highly adaptive and heritable, meaning that the system can tune itself to various viruses and pass it on to future generations. Ishino and colleagues first discovered the peculiar short repeats in the bacterial genome (CRISPR) in the 1980’s (1), and the CRISPR system was found to be present in about 40% of bacterial species (2). Other investigators independently established the presence of Cas coding genes (and hence, Cas proteins) adjacent to the repeats (3).

Both the components of the gene editing system are inherently present in bacteria. The bacterium leverages the CRISPR-Cas9 system elegantly to protect itself from an invading bacteriophage (viruses that attack bacteria). When a bacteriophage infects a bacterium, it gains control over the bacterial system by injecting its DNA into the bacterium. The bacteriophage’s DNA is then converted into proteins and the virus hijacks the bacterium, using it as a protein-making factory to make more of the virus. Bacteria impede viral takeover by producing RNA from the evolved and pre-existing CRISPR cassette assembled in the bacteria over years and years of evolution, that target the DNA of the bacteriophage only and corresponding Cas9 enzymes cleave the RNA-DNA hybrid. Unlike a one-time affair, the CRISPR system “remembers” parts of the bacteriophage DNA, thus generating “memory.”

Advancements in polymerase chain reaction (PCR)and genetic testing opened a treasure trove of information not only with respect to heredity and DNA fingerprinting, but also the genetic cause of diseases. If a faulty gene is the cause of the diseases, could scientists fix the gene?

Research in the field of genome editing was stalled until the advent of CRISPR-Cas9 technology, as all therapy before CRISPR-Cas9 lacked specificity and precision leading to detrimental side effects. The CRISPR-Cas9 system exquisitely solved the issue of specificity and precision. Certain factors affecting the efficacy of the CRISPR-Cas9 gene editing system include gene target designs and delivery systems. CRISPR-Cas9 systems target ~23 base pair (bp) sequences and the human genome is 3.2 billion bp large. The target design must be specific for the disease gene and avoid overlap with other genes.

Delivery systems have advanced from using inactivated viral systems as delivery agents to now packing the CRISPR-Cas9 module in nanoparticles ensuring greater efficiency of the system and reduced off target immune system activation. However, squeezing all the components required for the system into the delivery platform is still a challenge.

Additionally, Cas9, CRISPR’s team player, could trigger an unwanted immune response, that may cause undesirable and untimely cell death as the human body recognizes the Cas9 protein as a foreign particle because of its bacterial origin. In the year 2020, Dr. Jennifer Doudna and Dr. Emmanuelle Charpentier won the Nobel Prize in Chemistry for “the development of a method for genome editing. They pioneered utilizing CRISPR-Cas9 system for gene editing and engineering.”

In just three decades after the discovery of CRISPR-Cas9 system, the mammalian genome was successfully edited. Five years later, the first clinical trial for the use of CRISPR-Cas9 in cancer immunotherapy took place in the US. Many clinical trials are now underway to leverage the CRISPR-Cas9 system to treat sickle cell disease, leukemia, thalassemia, lung cancer, esophageal cancer, HIV-1, and many other debilitating diseases which were previously presumed untreatable.

Since CRISPR-Cas9 edits the genome, it has been the topic of debate and controversy. CRISPR-Cas9 can be used to edit the germline genome which is heritable, meaning that the changes you make in the genome can be passed on through generations. It raises a huge ethical concern if parents could choose characteristics of their unborn child, thus denying the unborn child of its autonomy. Concerns and controversies about the CRISPR-Cas9 technology arise regularly and are a major bioethical hurdle, preventing the widespread implementation of CRISPR-Cas9 technology. Recent research from the Gootenberg group has established that the CRISPR system can be used to edit RNA (4). This system targets messenger RNA (derived from DNA) instead of the master copy, which is the DNA.

CRISPR technology targeting DNA works with Cas9 protein, however CRISPR that targets RNA works with another closely related protein Cas7-11. Early research shows that RNA CRISPR is highly specific, with minimal cellular toxicity. This system is similarly programmable as the CRISPR-Cas9 system, but has distinct advantages. As stated by the central dogma of biology, a copy of DNA is transcribed (converted) into one or multiple copies of RNA. The RNA is then translated (read) into proteins which are the functional units of individual cells. In most diseases, the proteins are either dysfunctional or present in excess. In some diseases like hemophilia, a blood clotting disorder, some amount of clotting protein proves to beneficial to the patient suffering from the disease over the complete absence of clotting protein.

Programmable RNA CRISPR can be potentially used to control the protein gradient and hence the disease outcome. RNA targeting might be preferred for these transient changes as its effects are temporary (targets RNA, leaving DNA untouched) and flexible but can still regulate protein expression. RNA CRISPR can be expanded to treat diseases without a genetic origin, expanding its utility.  Therefore, RNA editing may overcome the technical and bioethical challenges associated with traditional CRISPR-Cas9 approaches warranting further research in RNA-CRISPR technology.

References

1. Ishino et al; 1987; 10.1128/jb.169.12.5429-5433.1987
2. Godde et al; 2006; 10.1007/s00239-005-0223-z
3. Bolotin et al; 2005; 10.1099/mic.0.28048-0
4. Ozcan et al; 2021; https://doi.org/10.1038/s41586-021-03886-5

About The Author
Raksha Parthasarathy is a 4th year graduate student in the Molecular Immunology & Microbiology discipline in the Integrated Biomedical Sciences Ph.D. Program. She is working in the lab of Dr. Elizabeth Leadbetter studying the role RIP kinases play in innate like lymphocyte biology. She serves as the Secretary for the Student Government Association. See her student profile and Pipette Gazette feature “Raksha Parthasarathy: My first memory of science was the Science Express, a science museum on wheels“>> WISDOM has partnered with The Pipette Gazette to start a new column dedicated to amplifying women’s voices at UT Health San Antonio. If you are interested in participating, email Hannah Elam.