Gene Editing: How it Works?

Science knows about many genetic disorders. Moreover, thanks to #GWAS (genome-wide association studies), science even knows about the role of exact genes or a combination of genes in different chronic ailments, like diabetes, cardiovascular disorders, and more.?

It means that gene therapy or editing can not only help treat specific genetic disorders like sickle cell disease but may also help prevent and treat other chronic ailments. Thus, there is an urgent need to find a safe and effective gene editing tool that is good for clinical use.?

To date, a tool that could help edit genes in the human body was missing. So, one can readily imagine the challenge of creating such a tool that can penetrate the body, enter most body cells, and carry out genetic editing. Fortunately, now science has found such a tool, and it is called CRISPR.

#CRISPR is now in an advanced stage of testing, and #FDA has approved many #clinical trials using CRISPR. Thus, it is just a matter of time before we see its widespread use in clinical practice1.

How CRISPR works?

Perhaps the biggest strength of CRISPR is that it is a relatively simple tool. Moreover, it is something that is also present in nature, making it among the safer tools to use.?

Like many great inventions, it was discovered just by chance. It was discovered in 2012 when researchers were studying bacterial immunity. They were exploring how bacteria defend themselves from viral attacks.

In the study, they found that bacteria have a CRISPR-Cas9 complex. Once the bacteria are affected by any virus, bacteria can keep a copy of a part of viral genetic material (DNA). The next time when the same kind of virus attacks, bacteria can identify it using CRISPR-Cas9 and delete part of its genetic material. It uses the copy of #DNA to produce its mirror image or RNA, and CRISPR uses this RNA to identify the #viral DNA. CRISPR #Cas9 complex carries out the so-called double-stranded breaks (#DSBs ) in DNA. Since viruses cannot repair their genes, this results in the neutralization of virus2.

Here it must be noted that CRISPR proteins hold the information about the chunk of viral material, and this information is present as RNA, whereas Cas9 acts as a genetic scissor. Thus, Cas9 can search the viral genome and look for a part to be deleted based on the information provided by RNA with the help of CRISPR3.

Thus, researchers came up with an idea. What if they could substitute the CRISPR RNA with some other kind of RNA? As CRISPR RNA acts as reference material. And they were right. If CRISPR RNA is substituted, the Cas9 protein will search the DNA for the mirror image of that RNA and carry out gene deletion precisely at that point.

Now researchers understand that this is a unique tool with unlimited possibilities. For example, they can now readily use this tool to remove faulty genes, like those causing cancers, genetic disorders, and more. Moreover, unlike viruses, human genes can repair themselves. It means that once the CRISPR has deleted the part of the gene, the human cell initiates a repair mechanism and joins the separated parts. Thus, the idea search and cut tool came into existence.

Moreover, it is worth noticing that CRISPR is very precise, as it carries the RNA copy and thus ensures that only the part of DNA that exactly matches this particular RNA is deleted. Other parts of DNA remain unaffected.

CRISPR and gene knockout

As one can see that CRISPR uses reference RNA, and then Cas9 carries out double-stranded breaks (DSBs) in DNA or deletes the part of DNA. However, unlike viruses, in other multicellular beings, DNA can carry out the repair after deletion2.?

The cells use the so-called non-homologous end joining (NHEJ) pathway to join the two separated ends of genes. This causes a shift in the gene, and the remaining gene becomes non-functional. This phenomenon is called gene knockout.

CRISPR and gene knock-in

Until now, we have seen that CRISPR can delete or knock out part of the gene, thus making the whole gene inactive. But what if we need to edit the gene instead? Well, that is also possible with CRISPR. For this, one would need to insert the same length of sequence that was deleted from the gene.?

For this, CRISPR uses a special kind of tech, and it is provided with a DNA template or donor template. This ability to edit genes using Cas9 and sgRNA has been one of the most significant breakthroughs in science as it not only allows the silencing of faulty genes but also carries out their repair. Quite often, gene silencing is not an option. Instead, there is a need to repair the existing genes2.?

In this approach, new genetic information is inserted, which is made possible by homology-directed repair (HDR). This ability to edit genes has opened doors for so many opportunities.?

CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi)

Deleting gene sequences and replacing them with something new is amazing, but sometimes all that is needed is altering gene expression, and CRISPR can also do this. CRISPRa can upregulate certain genes, making them more active, or CRISPRi can suppress the activity of specific genes2.?

Epigenetic changes in gene expression rather cause many common health conditions. It means that CRISPR can also be used to treat less severe health issues. For example, it may be used to reduce inflammation, treat allergies, prevent disease progression, boost immunity, treat developmental problems, and much more.

To sum up, CRISPR is a gene-editing tool found in nature, and now science is exploiting it to treat different health conditions. It can help delete gene sequences, edit parts of genes, and more. However, these are still the early days of the technology, and scientists are still perfecting it, and thus it is pretty likely that we may see many more uses of CRISPR in the near future.

References

1.????CRISPR Clinical Trials: A 2022 Update. Innovative Genomics Institute (IGI). Accessed October 20, 2022. https://innovativegenomics.org/news/crispr-clinical-trials-2022/

2.????The Ultimate Guide To CRISPR: Mechanism, Applications, Methods & More. Synthego. Accessed October 7, 2022. https://www.synthego.com/learn/crispr

3.????Mahmoudian-sani MR, Farnoosh G, Mahdavinezhad A, Saidijam M. CRISPR genome editing and its medical applications. Biotechnology & Biotechnological Equipment. 2018;32(2):286-292. doi:10.1080/13102818.2017.1406823