Optimizing Prime Editing: Novel Genetics Against Life Threatening Disease

- By Rishikesh Madhuvairy

For decades, genetic diseases had been inevitable. It was the natural bane of human existence, occurring simply due to structural inadequacies in the human genome. However, one might think: “The human genome in itself is the building block of an organism. It’s the cement needed to consistently, and precisely construct and reconstruct the building of a living thing”. Not wrong in the slightest. As human beings, we have witnessed the dawn of our life to the current breaths we take, without even knowing what had constituted our development over so many ages. It was indeed the ability of our genome to store information that helps us grow; To uniquely identify us; and above all, to biologically define our existence. Although we believe that we are indeed the crown of creation, we are certainly not inherently perfect. In fact, the human genome is in practice, the most 'imperfect perfect' biological structure that exists in an organism. Gene material is composed in our DNA. When a mutation makes changes to that material, the instructions given to human cells are altered, likely for worse. The mutation can result ideally from a pathogenic intervention in the chemical structure of DNA proteins, or simply the wrong amount of genetic material itself, such as an abnormality in chromosome count. Such marginal changes to the genetic composition of a cell, tissue, or organ, gives rise to diseases like Anaemia, Down Syndrome, and Liver Fibrosis. The issue at hand is that needless to say, we as humans are incredibly weak, in the grand scheme of things. But as a species, we are also incredibly intelligent. And so what was the most earth-shattering breakthrough that curbed these diseases for the better? As many know, CRISPR. CRISPR is the pioneer of gene editing, and is contributing to forming the infant genomic industry for genetic engineering in the future. The scientists who developed CRISPR analyzed how bacteria used genetics to evolve and ward off viral infections, and replicated the same using technology, in human cells, to boost their immunity, engineer stronger hereditary traits, and an amalgamation of similar facets. But in order to gain a deeper understanding about CRISPR technology, we must understand the Cas9 enzyme. CRISPR Associated Proteins, or Cas, are the main protein structures used for genomic editing, arguably more fundamental in qualitative comparison to prime editing. The function of a protein that constitutes a strand of DNA, is dependent on the characteristics of its amino acids. DNA consists of multiple nucleotides, the contents of which are nitrogen bases that are reordered to make the nucleotide unique (Adenine, Guanine, Cytosine, and Thymine). A codon is a sequence of 3 or more nucleotides, that dictate the function of the amino acid making up the protein. Amino acids are uniquely added to nucleotide chains when a cell undergoes protein synthesis. This occurs when genetic instructions are separated and copied to a single strand RNA (the messenger), through the formation and translation of the amino acids. Every time large protein chains are formed, the end-to-end physical contacts that exist between linking amino acids is known as protein-protein interaction. Codons play a role here, as a unique type of codon, called the stop-codon will assemble connecting nucleotides to the main protein itself, and therefore provide the cell with instructions to not undergo protein-protein interaction. The hypothesis proven is that this effectively stops the synthesis of the DNA. How can is this incorporated into novel gene editing? The Cas9 enzyme optimizes codons to act as genetic scissors, and halt the copying of instruction sets for a forming DNA, subsequently breaking the chemical bonds between the linking proteins. The Cas9 enzyme would use base pairing features, to cut off matching nucelotides with RNA, thus allowing the remaining protein to chemically bond through protein-protein interaction. This changes the structure of the DNA, and therefore changes the behavior and the instructions assigned to the cell. Therefore, this means that overall, the conclusion drawn is that CRISPR technology can recode a sequence of cell instruction sets. By changing the protein’s characteristics, the cell’s entire nature changes. This is the very same endonuclease present in genetically evolved bacteria. Thus, if they can perform these operations and minimize the risk of a viral disease, then it is worth hypothesizing that those genomic traits are reproducible in human beings, under a specific trial selection of varying phenotypes. All we need are the scientific foundations of genetic instructions. In some sense, the fundamentals of the same are just like binary data on a computer. Current CRISPR technology has rather been speculated to be “iffy” nonetheless. There have been several incident biomedical reports stating different allegations on CRISPR engineers, stating that it is “unethical”, with unintended consequences. There is still no clear line drawn on the extent to which this technology can be used, which opens up possibilities of intentional mutations to living cells, and genetic blending for strong super-human genes. It’s the closest we’ve come to supernatural. Introducing prime editing: A novel subset of gene editing that helps reform the discoveries scientists have made about Cas9. Prime editing specializes in guiding the Cas9 enzyme to the right genome for rearrangement of nucleotides, as well as serving as an RNA template that usually copies sequences into a genome, in regular CRISPR editing. CRISPR prime editing is more technologically advanced than CRISPR genomic editing, as, in simple words, it allows for more precise and adaptable genetic changes. This reduces the risk of an artifically induced genetic disease, and is therefore more remedial in nature.

How does our existing knowledge of Cas9 go hand-in-hand with prime editing? Well, there is a special element in prime editing that makes its genetic editing process a lot more accurate than conventional CRISPR genome editing, and that’s the prime editing guide — pegRNA. The Cas9 protein serves the same function as before: breaking down DNA strands and repairing them with a different genetic structure. However, pegRNA acts as a “guide” RNA that directs the Cas9 protein to a specific location in the genetic composition of the cell, enabling the DNA to be reshaped in a manner that targets a very exclusive part of the strand, that is prone to disease. The pegRNA oversees this through the reverse-transription of the RNA strand (often done through a component of the endonuclease substrate, called the transcriptase). Prime editing does not involve gene copying, as one can see above. Instead, it involves single-strand RNA production. This is much quicker and more dependable than general CRISPR editing, as it opens up the scope for minute insertions, deletions, and alterations to the DNA’s nucleotide structure. Genomic editing could still be used directly to a much larger/broader part of the DNA strand, therefore manipulating genes possibly in a dangerous way. Hence, prime editing has played a substantial role in this day and age, to reduce the repetition of unnecessary cycles of genetic duplication, and instead, use precise modifications on existing genomes. This contributes to prime editing technology being relatively cheaper as well. A normal pegRNA in prime editing would require only 1 pegRNA strand for a precise protein synthesis, wheras CRISPR needs multiple. Prime editing has been an innovative breakthrough not only for pioneering a new pathway of discovery in multiple fields, but for redefining evolution as we know it. The capabilities of creating viable products out of genetic engineering in prime editing are endless, and the investments into these global healthcare research projects give valuable insights on how important prime editing is to major companies furthering its cause in the quarternary sector; The cause of strengthening the human genome against diseases, bearing a minimal risk. Prime editing is currently being used by highly reputed genomic technology firms, incdluing GenScript, CRISPR therapeutics, and Beam, among others. The reason for this is the change in consumer/societal attitudes towards revolutionizing genetic healthcare in the biomedical industry. Prime editing is being used all over this industry, from treating the central nervous system of a Huntington’ s Syndrome patient with gene repair, mitigating the impacts of Klinefelter’s Syndrome on middle-aged adults exposed to the imminent risk of rheumatoid arthritis, or even strengthening lymph-nodes against microbial contaminants in chemically toxic air. If these are the scientific breakthroughs that prime editing is providing us in the 21st century, then it goes without saying that the future applications of prime editing are so far reaching, and yet uncertain. Currently, one specific future endeavour that I’d like to cite is cancer. The ability to reverse mutations of cancerous tissue is therapeutically efficient, and moreover a step towards curing such a disease. Using prime editing techniques to alter or reverse the mutations of cancer cells, the same concepts, if built on further by engineers, could establish a solution to cure cancer as a malignant disease altogether. Similarly, there are numerous other possibilities where people could be saved from autoimmune diseases just through one pair-up in their genes. CRISPR Prime editing, if publicized to the research world scientifically speaking, can help achieve such an oustanding collection of goals. At the end of the day, as the infinite fabric of knowledge unfolds over time, we can only conceptualize, and then anticipate that the story and technicality of prime editing simply goes to show us that the more we discover, the more that is left to discover. If used to its purpose, with accuracy, precision, non-duplicacy, delicate gene alterations, protein synthesis, and enzyme locating agents, prime editing would emerge as the newer technology of today’s world. With one exact nucleotide altered, an entire bacterial disease could harm you negligibly.