Behold the Revolution Heralder: CRISPR-Cas9

Jennifer Doudna (left) and Emmanuelle Charpentier (right) share the 2020 Nobel chemistry prize for their discovery of the game-changing gene-editing technique CRISPR-Cas9 system.

Precision, Accuracy, Speed

The 2020 Nobel prize in Chemistry was awarded to Jennifer Doudna and Emmanuelle Charpentier for the wonderful gene-editing tool- CRISPR-Cas9. CRISPR is actually a natural system in archaea and bacteria which helps fight invaders like viruses by chopping up their DNA and storing that DNA snippet as a part of the host’s own genome in the Locus of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR).

Let’s start with the basics. The base foundation of DNA ( DeoxyriboNucleic Acid) is a sugar base like ribose attached to a phosphate group and a nucleotide. Adenine(A), Thymine(T), Guanine(G) and Cytosine(C) are four options for nucleotides that form the crux of the entire living world.

Now let’s discuss some basic chemistry. A hydrogen bond is formed between Hydrogen and an electronegative element( loves to gain electrons) like Fluorine, Oxygen or Nitrogen, wherein these elements try to seize Hydrogen’s electron, which brings those groups closer as a result of partial delocalization, thereby stabilizing the overall complex. Adenine binds to Thymine via two Hydrogen bonds, while Guanine binds to Cytosine via three Hydrogen bonds. This is known as Watson-Crick base pairing.

Now, what exactly is a palindromic sequence? It is a nucleic acid sequence in a double-stranded DNA or RNA( RiboNucleic Acid) molecule wherein reading in a certain direction on one strand matches the sequence reading on the complementary strand.

For example, GAATTC is a palindromic sequence because if we look sequentially from both the endpoints, G is complementary to C, A to T and again A to T. This means that this sequence can base pair with itself, forming a kind of hairpin loop-like structure.

The CRISPR locus has several short palindromic sequences known as CRISPR spacers which are regularly separated with DNA sequences known as CRISPR repeats. When transcribed, i.e. when those DNA sequences are converted to RNA, which is the first step towards protein formation, these viral, chemical mugshots are encapsulated as RNA (range varies, usually 17 to 21 base pair long) in CAS ( Crispr ASsociating) proteins which verify the complementarity of those ‘guide’ RNA sequences with any DNA matter lying around. After successful verification, these molecular scissors chop up precisely at a particular sequence point. The chopping is done upstream, i.e., towards the 5’ end, from a region known as Protospacer Adjacent Motif or PAM consisting of three nucleotides- N(any nucleotide)GG. The presence of this sequence tells the bacteria that this is some foreign DNA as the bacteria’s own genome is devoid of it. This way, the second time the bacteria encounters a similar strain of invader, it would recognize it via the CAS proteins that would then proceed to chop up the invader’s genome.

An overview of a typical CRISPR locus

CRISPR spacers are the places where viral DNA are stored. They are typically 32 base pair long. CRISPR repeats separate CRISPR spacers and are typically 21 base pair long.

Cellular Swiss Army Knife

CRISPR is so much more than just molecular scissors. Numerous applications for CRISPR have come forward in recent times.

Some of them are listed here:

Neutralizing antibiotic resistance

The Cas protein receives a specific signal to facilitate the chopping up of the bacteria’s own genome so that it cleaves the antibiotic resistance genes of bacteria with precision. This special signal in the form of DNA is stored in a plasmid (extrachromosomal self-replicating DNA) which is introduced through a bacteriophage (bacteria-targeting viruses). As soon as the relevant genes are knocked off, the antibiotics can be administered, which effectively eliminate the bacteria. This way, CRISPR can be used to counter the menace of the ever-evolving antibiotic resistance to our drugs , which constitutes a huge problem while treating bacterial diseases in both plants and animals.

Quick and efficient testing

Cas proteins are of different types. Some target just the specified region of DNA while some chop the same along with any other regions of DNA localized near the site. These destructive Cas proteins cause collateral cleavage. These can be used to quickly and efficiently report whether a particular section of disease-causing DNA like that of a virus or bacteria is inserted in one’s genome after infection. After getting one’s DNA sample via nasal swab or a spit sample, PCR( Polymerase Chain Reaction) is carried out. PCR helps to amplify the desired DNA fragment. Subsequently, destructive Cas proteins along with a reporter gene are added to the PCR product mix. A reporter gene fluoresces when any of its parts is cleaved. So, if the Cas proteins have been trained to cut the infectious DNA part with collateral cleavage, they will cleave the adjoining DNA fragments of the reporter genes, too and thus cause the entire test tube to fluoresce. This forms a quick yet very easy test to identify whether someone has an infection of any sort.

Xenotransplantation

Xenotransplantation is the method of transplanting living tissues, cells or organs from one species into another. Pigs are currently thought to be the best candidates for organ donations. Retroviruses found in the DNA of animals like pigs tend to insert into the host genome, which can cause potential cancerous mutations and myriad immunity issues. Thus, programming the CRISPR system to cut specific portions of the pig DNA results in a much lesser probability of those retroviral portions affecting us humans after accepting the transplant. Embryos created from these modified cells and placed into surrogates create ideal donor animals with inactive viruses.

Ethical concerns and challenges

CRISPR Cas9 is an evolving technology with limitless scope. Over 5000 papers were published on CRISPR in the year 2020 alone! Cas9 is a type of nuclease which cuts the regions where the guide RNA tells it to. This means that methods to play around with genes have become more precise and reliable than ever before. But if not within ethical constraints, any promising technology can quickly become a controversy — and CRISPR is no exception.

The Controversial Germ-line designer babies

He Jiankui, a scientist at the Southern University of Science and Technology, Shenzhen, China, used CRISPR to disable the CCR5 gene in human embryos during IVF ( In-Vitro Fertilization). HIV ( Human Immunodeficiency Virus) uses the CCR5 gene to allow itself access into the human body. After disabling this gene, He aimed to create HIV resistant human babies.

In November 2018, the first gene-edited twin girls- Lulu and Nana, were born. The father of the girls was HIV positive, and so the couple agreed to this experiment. He’s intentions were undoubtedly good though the approach used was considered a bit too extreme and premature. CRISPR isn’t perfect yet. A lot more research needs to be done before we can perfect embryo editing. Even if we could, there are many ethical questions regarding germ-line editing- affecting directly the course of human evolution. He Jiankui couldn’t reproduce the required mutations to their entirety, and now the two girls are stuck with unknown CCR5 mutations for the rest of their lives. The ideal motto should be to make people better, not better people. If not controlled within limits, this juxtaposition of unnatural selection with random off-site mutations could prove fatal for our future.

All these points just depict one thing — CRISPR-Cas9 is an extremely new technology. We don’t yet have enough research done to predict the consequences or effects of a particular insertion or deletion accurately, especially in the case of germ-line cells. There’s this media hype about CRISPR being an easy and cheap technology. Well, the CRISPR-Cas9 system is definitely cheaper than the previously existing gene-editing techniques like Zinc Finger nucleases, TALENS or mega nucleases but unknown consequences due to mutations and off-target effects outweigh this point to a far greater extent. The regulations haven’t caught up with the technology yet and so a moratorium, i.e. a temporary suspension of activity in germline editing has been called forth by the leaders of this revolution in order to take some time and discuss its future usage. The parameters to be considered are as follows:

• First and foremost: The consent of the yet unborn baby.
• Social class affordability.
• The possibility of designer babies with enhancements and favourable traits leading to the creation of super-soldiers, or any indirect destructive use of this technology, almost manifesting science fiction horror in real life.
• The most important aspect is that- just because we can, should we?

Concluding Remarks

Genetic engineering will inevitably dominate in the coming times, unleashing unthinkable possibilities for us. CRISPR-Cas9 promising to be a frontrunner in the prospective advancements in this field. As we do have a revolution in our hands now, what we do now will directly impact our future. This powerful weapon can pose many issues if not yielded in the right and controlled way.

Here are some of the videos I referred to while writing this introductory bit.