CRISPR-Cas9

Human body has been under constant surveillance since the existence of humanity, and even now, after all this time, there are a lot of mysteries buried in it. For example, nobody knows what causes headaches.

Our bodies can safely be assumed to be the most sophisticated and complex bit of engineering. The DNA(Deoxyribonucleic acid) inside our cells, is a double-stranded helical structure that carries the genetic information for the development, functioning, growth, and reproduction of all known organisms and many viruses. All this info is encoded in little things called genes.

Genes decide almost everything about a living being. One or more genes can affect a specific trait. Genes may interact with an individual’s environment too and change what the gene makes. Genes affect hundreds of internal and external factors, such as whether a person will get a particular color of eyes or what diseases they may develop. Some diseases, such as sickle-cell anemia and Huntington’s disease, are inherited, and these are also affected by genes.

If only we could alter our genes, right? As mentioned earlier certain DNA encodings(genes) are responsible for certain functions. Diseases like diabetes, cardiac-diseases, Alzheimer's, and many more are termed as genetic. So if the genes responsible for these diseases are modified using the CRISPR technique then it would be a cure for otherwise incurable diseases.

Imagine how good it would be, you could decide practically everything about your offspring while it's in the embryonic stage. But is it possible? If yes, then, to what extent? Let’s find it out.

The answer to the former half of the question is YES, it has been made possible by the combined efforts of Nobel Laureates Emmanuelle Charpentier and Jennifer A. Doudna. Compared to previous techniques for modifying DNA, this new approach is much faster and easier. This technology is referred to as “CRISPR/Cas9” and it has changed not only the way basic research is conducted but also the way we can now think about treating diseases.

What is CRISPR?

CRISPR is an acronym for Clustered Regularly Interspaced Short Palindromic Repeat. This name refers to the unique organization of short, partially palindromic repeated DNA sequences found in the genomes of bacteria and other microorganisms. While seemingly harmless, CRISPR sequences are a crucial component of the immune systems of these simple life forms. 

Just like us, bacterial cells can be invaded by viruses, which are small infectious agents. If a viral infection threatens a bacterial cell, the CRISPR immune system can thwart the attack by destroying the genome of the invading virus. The genome of the virus includes genetic material that is necessary for the virus to continue replicating. Thus, by destroying the viral genome, the CRISPR immune system protects bacteria from ongoing viral infection.

In short, Using CRISPR the bacteria snip out parts of the virus DNA and keep a bit of it behind to help them recognize and defend against the virus next time it attacks.

How does CRISPR-Cas9 work? Let’s find out.

The CRISPR-Cas9 system consists of two key molecules that introduce a change into the DNA. These are (a) a protein-based enzyme called cas9 and (b)a guide RNA.

Cas9 acts as a pair of ‘molecular scissors’ that can cut the two strands of DNA at a specific location in the genome so that bits of DNA can then be added or removed. While the guide RNA consists of a small piece of pre-designed RNA sequence (about 20 bases long) located within a longer RNA scaffold. The scaffold part binds to DNA and the pre-designed sequence ‘guides’ Cas9 to the right part of the genome. This makes sure that the Cas9 enzyme cuts at the right point in the genome.

The guide RNA is designed to find and bind to a specific sequence in the DNA. The guide RNA has RNA bases that are complementary to those of the target DNA sequence in the genome. This means that, at least in theory, the guide RNA will only bind to the target sequence and no other regions of the genome.

The Cas9 follows the guide RNA to the same location in the DNA sequence and cuts across both strands of the DNA.

At this stage, the cell recognizes that the DNA is damaged and tries to repair it. Scientists can use the DNA repair machinery to introduce changes to one or more genes in the genome of a cell of interest.

What are some applications of the CRISPR system?

Beyond applications encompassing bacterial immune defenses, scientists have learned how to harness CRISPR technology in the lab to make precise changes in the genes of organisms as diverse as fruit flies, fish, mice, plants, and even human cells. Genes are defined by their specific sequences, which provide instructions on how to build and maintain an organism’s cells. 

A change in the sequence of even one gene can significantly affect the biology of the cell and in turn, may affect the health of an organism. CRISPR techniques allow scientists to modify specific genes while sparing all others, thus clarifying the association between a given gene and its consequence to the organism.

Rather than relying on bacteria to generate CRISPR RNAs, scientists first design and synthesize short RNA molecules that match a specific DNA sequence—for example, in a human cell. Then, as in the targeting step of the bacterial system, this ‘guide RNA’ shuttles molecular machinery to the intended DNA target. 

Once localized to the DNA region of interest, the molecular machinery can silence a gene or even change the sequence of a gene. This type of gene editing can be likened to editing a sentence with a word processor to delete words or correct spelling mistakes. One important application of such technology is to facilitate making animal models with precise genetic changes to study the progress and treatment of human diseases.

Future?

It is likely to be many years before CRISPR-Cas9 is used routinely in humans. Much research is still focusing on its use in animal models or isolated human cells, to eventually use the technology to routinely treat diseases in humans.

Certain issues must be resolved before its application to the human genome. In most cases, the guide RNA consists of a specific sequence of 20 bases. These are complementary to the target sequence in the gene to be edited. However, not all 20 bases need to match for the guide RNA to be able to bind. The problem with this is that a sequence with, for example, 19 of the 20 complimentary bases may exist somewhere completely different in the genome. 

This means there is potential for the guide RNA to bind there instead of or as well as at the target sequence. The Cas9 enzyme will then cut at the wrong site and end up introducing a mutation in the wrong location. While this mutation may not matter at all to the individual, it could affect a crucial gene or another important part of the genome.

If all these complications are resolved, which is just a matter of time, this technique may prove to be a boon for millions of people, suffering from genetic diseases, who have lost hopes of living a perfectly normal life.

All this said, I now leave you with high hopes and a burning question.

What would be the ethical implications of genome editing? Would deciding the physiological properties of a human being as per one’s will be immoral?

Present your views in the comment section. Thank you for your time.