Prime Editing: Improving the CRISPR-Cas9 system

If we all thought that the CRISPR-Cas9 system was going to be the greatest achievement of science in the field of genetic engineering, we were wrong. Prime editing, the improved version of CRISPR, allows you to edit gene sequences in one step, and could potentially solve 90% of genetic diseases. What does it consist of? What is the difference between them? What could it be useful for? KEEP READING!
I think the CRISPR-Cas9 system has been, and is, one of the most recurring themes on this website and its social networks. However, and to put ourselves in a situation, we need to refresh a little the memory of everyone who is a little confused. CRISPR-Cas9 consists of 2 fundamental elements. First, endonuclease Cas9, an enzyme capable of double-stranded cleavage (DBS) on a DNA molecule. On the other hand, a guide RNA, an RNA molecule, of about 20 nucleotides, responsible for binding to Cas9 and guiding it (hence its name), to a specific area of ​​DNA. What zone? The complementary area to this RNA guide.

Therefore, and as we all know, we can design the RNA guide, decide what nucleotide sequence we put, to guide Cas9 to the area of ​​the genome that we are interested in editing. Once the cut is made, the cell in question has two options. One, “sand and glue” the ends of the break. Or two, use a mold to repair that cut.

If our mission is to interrupt a gene in order to “turn it off” (this is known as knock out), the first option is useful. Why? Because in this process the cell can eliminate or add nucleotides at random and without any sense, and the most probable result is the appearance of a STOP codon or the alteration of the reading frame. In summary? That gene is no longer functional, and the protein it codes for will never be synthesized. Prime_Editing_CRISPR_Cas9_Biotecnologia_Maria_Iranzo_Biotec

What happens if what we want is to correct a mutation that is affecting the functionality of that gene?
The second option. We need the cell to fix that double-chain cut but introducing the changes that interest us. In this case, in addition to Cas9 and the RNA guide, a third element is required, a template sequence. This sequence is, again, designed to our liking, so that the repair enzymes of the cell itself will use that exogenous template and edit the cut area of ​​the DNA.

And this is where Prime Editing appears to improve this second application of the CRISPR-Cas9 system. This new system will require three components. First, a Cas9 enzyme that only cuts one of the two DNA strands (called Cas9 nickase). On the other hand, a kind of RNA guide that works both as a guide and as a template. This molecule has been baptized as pegRNA (prime editing guide RNA), and is characterized by having, at one end, the Cas9 binding area and the guide that directs it to the area to be edited; and on the other hand, the mold that will be used to repair the breakage caused. In this way, the addition of an exogenous mold to the system is no longer required.

But where is the trick? In the third component of this new system, a reverse transcriptase, which is to be introduced conjugated to Cas9.

The transcription process, in case someone has forgotten, is the transformation of DNA into RNA. Then, with the translation, the RNA becomes the proteins that will perform the functions ordered by said DNA. This is the normal flow of our cells. What do you think a reverse transcriptase does? What its name indicates, carry out the transcription process but in the opposite direction: from RNA to DNA.

The grace of the system is that the reverse transcriptase used that RNA template that contains the pegRNA, transforming it into DNA, to repair the breakdown induced by Cas9. Wonderful isn’t it?

But how does this whole process happen?
When this entire system lands in a cell, the first thing that happens is that the pegRNA will carry Cas9 and the reverse transcriptase into its complementary area of ​​the genome. Once there, Cas9 will cut the complementary strand of DNA. This break will be repaired by reverse transcriptase using the extra tail of the pegRNA as a template. In turn, the cell’s own endonucleases will remove the old excess fragment.

If you have understood the process, now we would find ourselves with an unsustainable situation: the two strands of DNA are not complementary since one is edited and the other is not. This phenomenon is called mismatch. But rest assured that this system also takes care of this: the Cas9 itself will introduce a new cut in the unedited sequence, and in this case it will be the cell itself that will detect the break. The enzymes of this cell will use the previously edited strand as a template to repair the cut. The result? The two chains of the DNA molecule edited to our liking.

Surprising truth? What are the advantages over CRISPR-Cas9? Firstly, an exogenous mold is not required to repair the break since the mold is already incorporated in the system. On the other hand, as LLuis Montoliu points out: “Edits are obtained with less variability (less INDELs (insertions-deletions)) and with a smaller number of unwanted mutations in similar regions of the genome (off-targets) than those obtained with the traditional Cas9 ”.

Furthermore, it is a much more precise system to correct small mutations: which are usually responsible for genetic diseases. Sickle cell anemia, for example, is an inherited disease that is caused by a single nucleotide change. Instead of an adenine, it is a thymine that appears in a region of the gene that codes for β-globin, one of the hemoglobin subunits. The result of this minimal change? Hemoglobin has a lower affinity for oxygen and anemia appears.

Like this, there are many cases of diseases induced by small mutations. In this scenario, it is estimated that Prime editing could solve up to 90% of diseases of this type. And for the moment, he has already been able to successfully correct the mutations that cause sickle cell anemia, Tay-Sachs disease, and successfully complete the incorporation of a gene variant that protects against Creutzfeldt-Jakob disease.

Disadvantages? It does not allow modifying large fragments due to the size limitation of the pegRNA. And for the moment, the greatest efficacy has been observed only in certain types of human cells, having yet to be tested for use in in vivo models and other cell lines.

As we always say: for the moment, it only remains to wait for the investigations to advance. We will continue to monitor the evolution of this new revolution in the field of genetic engineering.

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