CRISPR Gene editing

INTRODUCTION

"Genes are the story and DNA is the language that the story is written in"

We live in a time where technology is accelerating exponentially. The Wright Brothers created the first aircraft back in 1903 and 44 years later Chuck Yeager broke the sound barrier aboard an aircraft. Add 22 years more and there we are, setting our first step on the Moon. These timelines are truly inconceivable.

A similar revolution seems to be quietly taking place in this century. About 70 years ago, DNA was first discovered, and within a very short time we have discovered the natural mechanisms which allow us to change the genetic makeup of organisms. This is CRISPR.

There have been gene editing technologies in the past , most notably-

• TALENs (Transcription Activator-Like Effector Nucleases)
• ZFNs (Zinc Finger Nucleases)
• Homologous Recombination
• Base Editing
• Prime Editing

1. The above have attempted to provide consistent results but their efficacy has not exceeded 50% in most cases. CRISPR on the other hand has been regarded as the most reliable method to edit genes. The technology is still being explored for various use cases as it is relatively newer than other methods.

CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. The impact of this technology is even wider than the abbreviation. Through this article we will understand how this technology works, its impacts and its challenges.

1. WORKING

CRISPR is a defence mechanism used by a class of bacteria named E.Coli .CRISPR-mediated mechanisms empower bacteria to combat viral intrusions with remarkable efficiency. The following steps outline the sequence in which the CRISPR-Cas9 system eliminates viral intrusions-

    1.1 Acquisition of Spacer Sequences: When E. coli encounters a new viral invader (bacteriophage), it captures a small piece of the invader's DNA (or RNA) and integrates it into its own genome. These captured sequences are called "spacer sequences." The R in CRISPR stands for Repeats which is a repeating DNA sequence between the spacer sequences which act as a marker for the position of the spacer sequences for their identification within the process.

1. E. coli stores these spacer sequences in a specific region of its genome known as the CRISPR array. The special thing about these repetitive sequences is their palindromic nature. Palindromes(the P in CRISPR) are words which are pronounced the same when read forward or backwards like the word "kayak". Similarly, the spacer sequences have a palindromic nature where the nucleases have the same sequence on both sides of the double helix.

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5. 1.2 Transcription and Processing: The CRISPR array is transcribed into a long RNA molecule, which is then processed into smaller individual RNA molecules, each carrying one of the spacer sequences. These small RNA molecules are called CRISPR RNAs (crRNAs). One can think of them like messengers to the Cas9 protein, which carry the message about the sequence of the viral DNA which is to be neutralized.

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8. 1.3. Matching and Target Recognition: In the event of a subsequent viral attack, if the E. coli encounters the same virus or a closely related one, the crRNA guides the Cas9 protein to the viral DNA (or RNA). The Cas9 protein functions like a pair of molecular scissors using nucleases to cut the viral DNA upon its identification.

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10. The purpose of identification of the viral DNA using the Tracer RNA is to make sure that the Cas9 protein is not eliminating any DNA sequence which belongs to the bacteria itself

1. 1.4. DNA Cleavage: When the Cas9 protein locates the viral DNA that matches the crRNA, it cuts the viral DNA at that specific site, creating a double-strand break. This essentially stops the potential translation of the viral DNA into a protein, which can affect the cellular functioning of the bacteria. Note that Cas9 is separating the DNA strands at an extremely precise location, which is right after the sequence matching. The cleavage itself is done using an enzyme called nuclease which is used to break the bonds of the sugar-phosphate backbone which keeps any DNA strand intact.

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3. 1.5. Cellular Repair Mechanism: When the viral DNA is cut, the cell's repair machinery is activated to mend the break. There are various possible ways in which the DNA can be repaired. One is direct recombination, where the the recombination enzyme tries to reattach the sugar-phosphate chain. Another way is insertion of a nearby strand of DNA for repair. there are multiple approaches the immune system can take, of which the two mentioned are the most common way.

1. 1.6. Immunity and Memory: Importantly, E. coli retains a "memory" of past infections by preserving the spacer sequences in its CRISPR array. This means that the next time the same or a closely related virus attacks, the bacterium can use the CRISPR-Cas9 system to defend itself more effectively.

This is the working of the CRISPR-Cas9 system.

2. Hacking the defence mechanism

The question now arises, how is this mechanism hijacked in order to edit genes as per human requirements?

This is done by making some changes to the process described in section 1 at different stages. Instead of a viral DNA, a target DNA is used which is to be modified.

1. 2.1 Designing the Guide RNA (gRNA): Researchers design a short piece of RNA called a guide RNA (gRNA). This gRNA is engineered to be complementary to the DNA sequence they want to edit. The gRNA can be considered as the messenger which will provide the address for the cleavage location in the target DNA sample.

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4. 2.2 Cas9 Protein: As discussed in section 1.4 the Cas9 protein is where the target DNA is to be dissected. It's loaded with the gRNA and directed to the target DNA sequence. Now since the gRNA is designed as per human specifications, researchers can define the exact location where the edit is to be made.

5. 2.3 Targeting the Gene: The gRNA-Cas9 complex is introduced into the target cells, often using methods like electroporation or viral vectors. These are methods used to invade a cell membrane for research purposes .

   
   
   

6. Once inside the cell, it searches the complex looks across the genetic sequence to find the particular target DNA sequence which is to be edited.

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8. 2.4 Cutting the DNA: When the Cas9 protein locates the target DNA sequence, it binds to the DNA and creates a double-strand break at that precise location. This break triggers the cell's natural DNA repair mechanisms.

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10. 2.5 DNA Repair: The cell repairs the DNA break using one of two primary repair pathways:

• Non-Homologous End Joining (NHEJ): This pathway is prone to errors and can introduce small insertions or deletions in the DNA sequence. This is not the ideal case we are looking for in the editing process
• Homology-Directed Repair (HDR): In this case, a DNA template with the desired genetic change is provided along with the gRNA-Cas9 complex. While in the repair process, the cell uses this template which has the related information for the new edit. This is the ideal case for making the edit.

1. 2.6 Gene Editing Outcome: Depending on whether NHEJ or HDR repair predominates, the gene can be edited in different ways:

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• In NHEJ, the gene may have random mutations or be disrupted.
• In HDR, the gene can be precisely modified according to the template which was engineered for making the desired edit in the genome.
  
  
  

1. 2.7 Validation: Researchers verify the success of the gene-editing process by analyzing the edited DNA sequence and assessing the functional consequences of the genetic changes.

To put it concisely, first the target location is identified and a gRNA strand is engineered. This strand is then passed to the Cas9 protein, followed by its introduction in the target cell genome by various methods. The Cas9-gRNA complex then locates the desired location and cleaves the target DNA, which is followed by a recombination process where the new DNA strand is introduced which becomes part of the target DNA during recombination.

3. Applications - 

Genes are the fabric which shapes our bodies, our behaviour, our intelligence, our entire being. Having the ability to alter this very fabric is fundamental to how we move ahead as a species. There are a multitude of applications of this technology -

3.1 Cure to genetic diseases: The very cause of a genetic disease is that a particular gene has mutated. Since CRISPR- Cas9 can edit genes, it has now become possible to cure such diseases given that we have enough information about the genes which are mutated in the first place. In some human trials, the genetic disorder of Sickle cell was cured.

3.2 Vaccine development: During the very recent Covid-19 pandemic, CRISPR was instrumental in the development process of vaccines. It helped researchers quickly analyse and modify genes of SARS-CoV-2 virus strains to develop potential vaccine candidates. Additionally, CRISPR helped improve the efficacy of vaccines and also helped to modify them for better temperature storage.

3.4 Anti-Ageing: There are some genes which directly affect the ageing process and can be modified in such a way that we could slow down or even reverse the ageing process. Some experiments we carried out on mice to validate this hypothesis and researchers were able to slow down the ageing process in mice. The mice became healthier and their lifespan increased significantly.

3.5 Designer babies: Since this technology is built to edit genes, it can be used to potentially select particular traits and edit genome in embryos. This is no more a theory but has been implemented on human embryos by Dr.He Jiankui on November 5, 2018 in China. T
he embryo was of twin girls in which Dr.He edited certain genes which are responsible for increased risk of HIV in humans. The twins were born with no genetic defects or deformations & have been living a relatively healthy life.

4. Challenges -

One can use nuclear fission to generate energy for all of civilization or annihilate entire continents using nuclear weapons. Ultimately, it is up to us how any technology is used. The same is true for CRISPR. One could use it to cure diseases but at the same time use it to build bio-weapons for mass destruction. 

The use of the technology isn't the only challenge here, but the lack of understanding of biology itself is a big hurdle which we would need to address in order to deploy CRISPR for particular applications.

Broadly, there are 2 types of challenges CRISPR faces-

4.1.Ethical challenges- With every new technology we are faced with many questions as a civilization. If we consider the use of CRISPR in editing human genes, we do not know the repercussions of introducing a new type of genome in the human gene pool, since nothing remotely similar has been done or studied in the past and could potentially destabilize the human population.

Another question which is very prominent to ask is, could CRISPR be used as a deterrent for wars like nuclear weapons were in the 20th century? It can be a very sensitive situation as we are not short of nuclear mishaps, a genetic one would likely be more destructive.

4.2.Technology limitations- We have sequenced the entire human genome which is over 3 billion base pairs long by the completion of the Human Genome Project in 2003. Even though we have sequenced the human genome, it is still unclear what certain parts of the genome do and how it affects the rest of the body. Hence, it becomes very difficult to decide upon the modification or deletion of certain parts of the genome for performing edits.

Another challenge the technology suffers is the existence of Homologous genes. These are genes where the sequence of the same gene affects 2 different aspects of the body. So for example, the same gene maybe responsible for eye colour and bone density. It then becomes difficult to change only particular aspects of the human body as they are not exclusive to each other but interrelated.

In conclusion, we are at the very cusp of a new technological revolution which can completely alter who we are as a civilization, how quickly we can adapt to changes, new methods to cure diseases, grow crops more efficiently, maintain our youth, design our next generation and possibly the engineering of the first super-humans in the literal sense. The ethical questions must be answered first and the technology applications must be closely supervised in order to maintain stability in the world. 

 " Technology is a useful servant but a dangerous master" - Christian Lous Lange