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From Bacteria to Breakthroughs: The Evolution of CRISPR

Updated: 5 days ago

In the unseen microbial world, bacteria had their own way of defending themselves against viruses. They didn’t have soldiers or weapons, but they had a smart system CRISPR-Cas. It was a sort of "genetic memory" that let bacteria recognize invaders (like bacteriophages) and destroy them by slicing up their genetic material.




At the heart of this defense system was a protein now world-famous: Cas9.

 

For years, this tiny molecular scissor quietly did its job until scientists discovered it and realized they could hijack this bacterial tool to edit DNA. The world of biotechnology would never be the same again.


The Cas9 Era: CRISPR 1.0


CRISPR Cas9 became the super hero of gene editing technology. In simple terms we could guide the Cas 9 enzyme to specific location in the gemone using a short RNA sequence that acts as a guide to reach the spot. Once there, cas9 would create a cut in the DNA, by using this method scientists could either delete a faulty genes or insert gene of interest.

 

This technology paved way for treating diseases that were once untretable like sickle cell anemia. Not only healthcare industry but also helped in development of genetically modifies crops that could resist drought.


This technology created a hope in striving cure for  Major life thretning diseases like

cancer, rare disorders, and even blindness accelerated.

 

This was no less than a revolution in the Biotechnological research



 

But like all technologies, Cas9 had its own limitations.


It was precise, but not perfect. Sometimes it cut the wrong spots which was not intended and this cut was considered as off-target effects, raising concerns about safety. Moreover, it could only cut DNA, which meant its uses were somewhat limited.


Scientists, always striving for better precision, didn’t stop there!!


CRISPR 2.0: The Birth of Base and Prime Editors


 

We all know that point mutation is common aspect encountered during DNA replication, althought the cells have different mechanism to correct them. Even with the different proofreading mechanisms some mutations still manage to escape the correction leading to point mutation.

 

What if I say , these point mutation can be corrected?

Yes you heard it right !!

CRISPR 2.0 Base editors are the latest upgradation of CRISPR technology


Imagine being able to change a single letter in the DNA like fixing a typo in a book. That’s what base editing did. No cutting, no chaos just a smooth correction.

 

Then came prime editing, often called the “search-and-replace” function of gene editing. Prime editors could not only correct a typo but also rewrite a sentence or insert a new one, all without breaking the DNA strand.

 

This was a gentler, more refined form of CRISPR, offering more control and fewer errors.




 


Prime editing components :


a.     Cas9 Nickase: Modified version of cas9 that create cut only on one DNA strand.

b.     Reverse Transcriptase (RT): Enzyme to re-write

c.     pegRNA (prime editing RNA): That guides the Cas9 to desiered location and consist of template that includes the RNA sequence of corrected base pair of gene, This is called as primer binding site to start the reverse transcriptase.

 

Step by step explanation of this mechanism:


  1. Step 1: Identify the gene having mutation and design the pegRNA with primer binding site.

  2. Step2: Use the cas9Nickase to create a break in one DNA strand.

  3. Step3: The attached pegRNA will use the primer binding site sequence and Reverse transcriptase to correct the gene.

  4. Step4: The newly synthesised gene containing the corrected sequence gets integrated into the genome as the cell repairs the nick.

  5. Step 5: The cell then corrects the other strand by the natural DNA repair mechanism.

 

Advantages of Prime Editing:


  • No need for donor DNA

  • No double-strand breaks (DSBs)

  • Precise editing (insertions, deletions, point mutations)

  • Lower off-target effects compared to traditional CRISPR


In summary, prime editing represents a powerful and precise gene editing tool that allows targeted DNA corrections without introducing double-strand breaks offering safer and more versatile solutions for treating genetic disorders.





Reference:


Anzalone, A. V., Randolph, P. B., Davis, J. R., Sousa, A. A., Koblan, L. W., Levy, J. M., ... & Liu, D. R. (2019). Search-and-replace genome editing without double-strand breaks or donor DNA. Nature, 576(7785), 149–157. https://doi.org/10.1038/s41586-019-1711-4

 

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