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Abstract: CRISPR Cas9 is one of the most promising treatment technologies for genetic diseases and many rare diseases. This article talks about the fundamentals of CRISPR Cas9 and its application in clinical trials for treating Sickle Cell Disease and further scope.
In broad terms, genetic engineering entails modifying the genome or parts of it using biotechnology. CRISPR Cas is one of the most rapidly developing technologies of genetic engineering, and it is based on the property of genes that provides adaptive immunity to microbes against phages and plasmids. In simple words, the genetic makeup of certain microbes showed sets of 29 nucleotide repeats withholding certain palindromic sequences, which can later form a hairpin structure, interspaced by 5 intervening 32 nucleotide nonrepetitive sequences, which were similar to pathogenic nucleotides. These sets were later classified as clustered repeat elements, which are present in 90% of archaea and >40% of sequenced bacteria. These clusters of significance are the basis of CRISPR, the acronym for Clustered Regularly Interspaced Short Palindromic Repeats. The protein Cas9 associated with this technology is an RNA- guided endonuclease that induces breakage at target sites on double-strand DNA. The palindromic sequences can form the hairpin structure when an external viral nucleotide sequence is incorporated into these clusters, marking the target site for the Cas protein to cause breakage and put the infection to halt (1,2).
In genetic engineering, three types (I–III) of CRISPR systems have been identified across a variety of microbial hosts. Each system comprises Cas genes, non-coding RNAs, and a distinctive array of repetitive elements (direct repeats) interspaced by short variable sequences called protospacers, together forming the CRISPR RNA (crRNa). In the DNA target, these protospacers are always correlated with a variable Protospacer Adjacent Motif (PAM), which later plays a crucial role in identifying the nuclease binding site. The Type II CRISPR system is one of the most distinctive and widely used, consisting of the nuclease Cas9, the crRNA array that encodes the guide RNAs, and an auxiliary trans-activating crRNA (tracrRNA) that divides the crRNA array into separate units, each of which contains a 20-nt guide sequence and a partial direct repeat. The 20-nt guide sequence directs Cas9 to a 20-bp DNA target via Watson-Crick base pairing by separating the two strands of DNA, facilitating the cutting of DNA at the target location. Consequently, the cell’s DNA repair mechanism repairs the broken strand, at which point the base pairs are added, or the whole repair mechanism can be manipulated for various genetic requirements (3). More recently, scientists have been able to modify the Cas9 nuclease to associate specific enzymes which create mutation of a single base in the target sequence, which may help in turning a disease-causing gene into a healthy one or may induce a stop codon to turn the gene expression off (4).
SCD, most prevalent in Sub-Saharan Africa and quite common in parts of South America, the Middle East, and India, is an umbrella term encompassing a group of lifelong inherited diseases (including sickle cell anaemia (SCA), HbSC, and HbSβ-thalassaemia) caused by mutations in the gene encoding the haemoglobin subunit β. The disease is characterized by the inability of Erythrocytes to carry oxygen resulting in the HbS (sickle cell haemoglobin) polymerization, which alters the lipid bilayer and proteins of erythrocytes leading to the formation of abnormal sickle-shaped erythrocytes. The oxygen affinity of these erythrocytes is further reduced by 2,3-diphosphoglycerate (2,3-DPG), a glycolytic intermediate that is physiologically present at very high levels in sickle erythrocytes, which eventually increases the rate of formation of Sickle erythrocytes. These Sickle-shaped erythrocytes are highly unstable along with an extremely reduced lifespan of 10-20 days. Also, they tend to be more adhesive than normal erythrocytes, causing vaso-occlusion and reducing blood flow depriving tissues of oxygen, creating an inflammatory response (5). As a result, people with SCD suffer from bouts of extreme pain and elevated risks of heart attack and organ failure.
Talking about advancements of CRISPR in Sickle cell disease (SCD), scientists have been able to treat patients under an ongoing trial to a large extent by gene editing, leading their bodies to produce foetal haemoglobin. In a recent trial headed by Dr Haydar Frangoul, the subject Jimi Olaghere was first treated with drugs to release his stem cells into his bloodstream, which were then harvested. These stem cells were then genetically engineered by CRISPR Cas9 technology. The BCL11A regulator is responsible for shutting down the production of foetal haemoglobin, which is the build-up of 2 alpha and 2 gamma subunits, making it physiologically advanced in its oxygen-carrying capacity during gestation, after birth. Using this technology targeting BCL11A, the production of foetal haemoglobin was turned back on. To eliminate the stem cells producing abnormal erythrocytes, Jimi was treated with chemotherapy, and then the engineered stem cells were transferred back to Jimi’s body. These stem cells started producing normal healthy foetal haemoglobin, typically downsizing the overall painful symptoms of SCD and beta-thalassemia. Although it is a highly exhausting procedure, it has shown promising results in many other subjects suffering from SCD and beta-thalassemia (6, 7).
However, CRISPR is not only limited to basic genetic engineering. Numerous trials are in progress using CRISPR for unconventional techniques too. Some of these include (i) associating transcription activators to guide RNA, which bind to target DNA to increase the transcription by bringing RNA polymerase too, (ii) gene silencing by similarly inactivating transcription, and (iii) attaching fluorescent proteins to the Cas complex to locate particular DNA sequences in a cell (4). Considering these advancements, it wouldn’t be a wrong conclusion to say that CRISPR has an immense potential to treat rare genetic disorders as well as remarkably revolutionise research as a whole.
REFERENCES
Hsu, P., Lander, E., & Zhang, F. (2014). Development and Applications of CRISPR-Cas9 for Genome Engineering. Cell, 157(6), 1262-1278. doi: 10.1016/j.cell.2014.05.010
(2022). Retrieved 13 March 2022, from https://www.youtube.com/watch?v=ezfwqmKC9Uc&t=202s
Ran, F., Hsu, P., Wright, J., Agarwala, V., Scott, D., & Zhang, F. (2013). Genome engineering using the CRISPR-Cas9 system. Nature Protocols, 8(11), 2281-2308. doi: 10.1038/nprot.2013.143
(2022). Retrieved 17 March 2022, from https://www.youtube.com/watch?v=4YKFw2KZA5o
Kato, G., Piel, F., Reid, C., Gaston, M., Ohene-Frempong, K., & Krishnamurti, L. et al. (2018). Sickle cell disease. Nature Reviews Disease Primers, 4(1). doi: 10.1038/nrdp.2018.10
Sickle cell: ‘The revolutionary gene-editing treatment that gave me new life’. (2022). Retrieved 17 March 2022, from https://www.bbc.com/news/health-60348497
(2022). Retrieved 17 March 2022, from https://www.youtube.com/watch?v=mQ8Ola_C5po&t=66s
About the Author
Author: Jayanti Chhillar Bio: Jayanti Chhillar is a final year student, studying B.Sc. (H) Biological Sciences at Sri Venkateswara College, University of Delhi. As a curious kid, she always found intricate cellular and molecular mechanisms and their biochemical pathways fascinating, following which, she aims to pursue higher studies in the field of Molecular Biology and Cancer Therapeutics. Stem cells and Induced Pluripotent Cells is another promising domain of research that she finds extremely captivating and innovative. Inquisitive about, intracellular signalling and functioning, she wants to explore ways by which they can be manipulated for clinical purposes, and consequently develop better medications and contemporary treatments for Cancer.
She loves to interact with like-minded people, understand their perspectives and discuss and learn more about science in general, which is primarily why she’s eagerly looking forward to joining the TSV team.
Editor: Chiya Goyal
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