A Breakthrough in Modern Genetic Engineering, Introducing CRISPR

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What is CRISPR?

Just as humans have immune systems to protect us against pathogenic microbes, bacteria have also evolved mechanisms that induce an antiviral response. Upon initial exposure to a virus, some bacteria will collect pieces of the viral DNA and incorporate it into its own genome, creating Clustered Regularly Interspaced Short Palindromic Repeats, also known as CRISPR arrays.

Secondary exposure to the same virus will then initiate an antiviral response in which the CRISPR array is used as a template to create an RNA molecule, called a short guide RNA (sgRNA), which will bind at complementary sites to the viral DNA. “Molecular Scissor” proteins called CRISPR Associated (Cas) proteins are then recruited to cut the viral DNA, thereby inhibiting viral DNA replication, gene expression, and pathogenesis.

This mechanism was discovered by scientists researching bacteria in yoghourt who took note of this unique mechanism.

In recent years, scientists have developed a method in which to use the same CRISPR machinery to make changes to any DNA molecule. Using a programmable and synthetic sgRNA and the Cas9 protein (or any Cas protein), it is now fairly simple and inexpensive to edit DNA in a living organism.

How does it work?

In a laboratory setting, a short guide RNA is synthesised to be complementary to the DNA sequence we would like to edit. This sgRNA then couples with the Cas9 protein and guides it to the target DNA sequence. The protein is then able to cut the DNA at the target sequence and the cell’s natural DNA repair systems will “reseal” the double helix.

To simply inactivate a gene, one sgRNA and Cas9 protein can be used resulting in a single cut spanning both strands of the DNA sequence. This cut is then resealed by the cell’s DNA repair enzymes however, a few nucleotides (DNA building blocks) are excluded from the repair. This process results in a DNA sequence slightly different from the original, thereby inactivating or silencing the gene of interest.

To fully delete a gene, two sgRNAs and respective Cas9 proteins are used, each flanking the beginning and end of the gene. The Cas9 proteins will each make a single cut spanning both DNA strands, and the section of DNA between the two cuts will be removed. The remaining DNA surrounding the gene of interest will then be ligated together using the cell’s natural repair mechanisms.

Lastly, the CRISPR/Cas9 system can be utilised to replace an old genetic sequence with a new sequence. In this instance, two sgRNAs and two Cas9 proteins are used to resect a gene, as described previously, however this method incorporates an additional DNA template. The DNA template will be ligated to the neighbouring DNA strands in place of the old gene, resulting in a completely new gene or sequence.

What are the potential applications?

CRISPR/Cas9 and related technologies are continuously improving to become more accurate, simpler, less expensive, and more robust in their applications.

Currently, CRISPR is widely used in research laboratories across the globe. With this tool, researchers can edit their model organisms to be missing a gene, have several copies of a desired gene, silence a gene, and more. These changes allow them to investigate the roles of genes, including the implications of their deletion or overexpression.

Huntington’s Disease – New Hope

The most obvious future application for a gene editing tool as powerful as CRISPR is the potential curative power for single gene mutation diseases. An example of one such disease is Huntington’s Disease, a hereditary illness caused by a mutation in the HTT gene. This mutation is known as dominant, meaning that an individual needs only to inherit one copy of the mutated gene to develop debilitating symptoms such as loss of motor control or rapid cognitive decline.

Using CRISPR/Cas9 technologies, the mutation in a patient’s HTT gene could theoretically be removed or replaced, potentially curing the patient of their disease. This theory applies to any disease causing single gene mutation and has the potential to save and improve countless lives.

Unborn Embryos

Sometimes referred to as “designer babies”, the scientific community has discussed the potential genetic editing of embryos to impart a specific set of traits upon the person when they are born. For example, an embryo could be specifically edited to ensure the baby will have green eyes, brown hair, and perhaps a higher potential for athletic abilities. This is, of course, just a thought experiment at this point since the genes related to each human trait have not been fully elucidated, and because there is currently a moratorium on the genetic editing of humans. If the moratorium is ever to be lifted, it would most likely be to allow for the editing of harmful genetic sequences, such as mutations in HTT, in unborn embryos or adults living with the disease.

HIV

Another exciting potential application for CRISPR genetic editing technologies is the treatment of retroviruses, such as Human Immunodeficiency Virus (HIV). HIV is a virus that, if left untreated, will progress to Acquired Immunodeficiency Syndrome and kill the infected individual. Without getting into the nitty gritty of retroviral life cycles, it is important to note that HIV and similar viruses integrate their genome into the genome of the host’s immune cells.

This means that it is nearly impossible for the host immune system to clear the viral infection because the immune cells themselves become a production factory for new virus particles. In the future, CRISPR technologies will hopefully have the power and accuracy to remove the HIV genome from host cell DNA to clear the infection. This would be the first cure to HIV, having the potential to save thousands of lives.

Similar to their role in bacteria, CRISPR arrays and their respective Cas proteins may be a useful tool in vaccine development. Certain vaccines, called recombinant vaccines, function through the insertion of a viral gene into the genome of another, less dangerous virus. The viral gene that is inserted will encode for a protein that will trigger an immune response and development of antibodies against the target viral antigen, thereby vaccinating the individual against the virus.

The processes of adding antigen genes and removing pathogenic genes can be difficult and cumbersome at times however, CRISPR technologies may be able to assist in the process. Using CRISPR, scientists can now easily delete or add any viral genes into the recombinant genome. This process will allow for safer recombinant vaccines that can vaccinate against many strains of a virus or viral antigens.

CRISPR has already, and will continue to, change the face of molecular biology and biomedical research.

With the power to change the blueprint of our very essence, this technology has the potential to drastically improve, or save, the lives of countless individuals.