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Revolutionizing Genome Editing with CRISPR
Mark Behlke, CSO, Integrated DNA Technologies
Functional genomics is the area that interfaces between the use of synthetic oligos or nucleotides and living cells in a way that it changes the genome or changes gene expression and includes anti-cells, RNA interference, splice-switching oligos, and genome editing. Even though genome editing has existed for years, it has caught the trend recently due to Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR). The traditional method of genome editing includes zinc-finger proteins, zinc-finger nuclease, talens, and megaTALS. These older technologies required protein engineering for every different target site and scientists had to create a new protein every time they changed the site, which took six months of work to try a new site. But in CRISPR, the protein component stays the same and only the Guide RNA changes, which can be mass produced. Consequently, thousands of sites can be simultaneously monitored and changed as desired. This new method has considerably reduced the inconvenience and the time lag associated with site changes. Now, the barrier to working with the genome editing technology has been vastly reduced and has opened up new possibilities towards correcting ailments through gene therapy.
A genome editing experiment with CRISPR requires CAS 9 nucleus and a gRNA in the cell. The editing experiment can be carried out by two methods—firstly, CAS 9 nucleus and a synthetic single gRNA is integrated into a plasmodium expression construct, which is then placed into a cell. A CAS 9 and a gRNA would be made and then the editing would occur.
Though it can take a long time for these technologies to create useful drugs, do not give up the quest—keep working on it
The second method in the natural setting occurs in bacteria with CAS 9, and two actively separate gRNAs. First, a long tracrRNA, which binds and mediates activation of the CAS 9 from an inactive to an active conformation, and the second is crRNA, a short target specific RNA. A crRNA is subdivided into a target specific domain with 20 bases targeting the genomic DNA that is supposed to be sliced and the other segment binds the tracer. At this point, the CLE base sequence will be targeting locus in the genome that the scientist wants to edit by getting CAS 9 and the RNAs into the cell which will make a cleavage at the required site. The cleavage of that then gets healed by different repair mechanisms.
The Plasmodium expression though used extensively can create a lot of unwanted side effects as in some cases the plasmodium itself can get integrated into repair sites or other sites of the genome and create off targets, a big problem in polymerase chain reaction (PCR). At Integrated DNA Technologies’ (IDT), our team has generated HiFi nucleus with the intention of finding mutants that retained activity as ribonucleoprotein (RNP). Additionally, we effectively created a genetic screening system of mutant genes in bacteria. Wherein we had a toxin gene that had an on target site in it and a necessary gene that included an off-target site and removing either of the genes will result in the death of the cell. Consequently, the IDT solution when delivered as an RNP reasonably forced a genetic competition with these mutants where the scientists have to edit on-target and not off-target sites.
Prospective Use of CRISPR in Various Field Studies
In the early phases of ex vivo CRISPR development, the therapeutic measures can be utilized in any hemoglobinopathy treatment such as sickle cells, beta-thalassemia and anything related to bone marrow production that can be removed from the body, modified, and then re-infused back into the patient. Hematopoietic stem cells, T cells, and other immune cells are the areas editing can be applied to. Apart from that, the liver has a great prospect in gene editing as scientists over the years have created the most advanced delivery technologies to transport large molecular drugs for it.
There is a grand future for nucleic acids in medicine especially in the diagnostics area where DNA is being used to determine the dominant and dormant diseases. Cancer treatment is also being revolutionized by gene therapy and opening up research opportunities to eliminate carcinogenic cells. But taking into account the historical context of several pharmaceutical companies, the one message I want to share is that though it can take a long time for these technologies to create useful drugs, do not give up the quest—keep working on it.