With the advent of genome editing technology, scientists can now directly target and alter a  living organism's genomic sequences. It has made it possible to create more precise cellular  and animal models, which has increased understanding of the genetics underlying human  disease. Its potential is outstanding in a wide range of domains, including biomedical research,  applied biotechnology, and basic research. 

The most common tools for gene editing up until 2013 were engineered nucleases, such as  zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). However, the rate of advancement in the sector was constrained by their relatively low editing  efficiency and lengthy development timeframes.^[1] 

The science of genome editing was revolutionized in 2013 with the discovery of clustered  regularly interspaced short palindromic repeats (CRISPR)/Cas-associated nucleases, which  are derived from a bacterial adaptive immunological defense mechanism. Short guide RNA  (sgRNA) is used by the CRISPR/Cas9 system to control Cas9-mediated cleavage and donor  HDR template insertion. A new era of genomic engineering has been brought about by the  ease of use, adaptability, and high degree of adjustable nature of RNA design to retarget Cas9.  This approach offers notable advantages over ZFNs and TALENS. In order to improve  efficiency and lessen off-target effects, base and prime editing, as well as modified or  alternative CRISPR nucleases (such as Cas12 and dead Cas9), are being researched in the fast  developing field of gene editing.^[2]

When comparing the off target effect is seen lower in ZFN, TALEN and high in CRISPR/CAS this  becomes challenge in utilizing rather less time consuming gene editing technique and reason  behind this is the target site of action which is DNA in case of CRISPR/CAS they can sometimes  localize to unintended sites with sequence similarity to the on-target site. When a CRISPR-Cas  system localizes to unintended sites and performs its programmed function there, this is an  off-target effect. The off-target effects occur when Cas9 acts on untargeted genomic sites and  creates cleavages that may lead to adverse outcomes.^[3]

 Treatment for cancer has been transformed by the gene engineering of T cells to create novel  cancer immunotherapies such chimeric antigen receptor (CAR)-T cell therapy. While the  clinical success rate of current autologous CAR-T immunotherapies is excellent,  improvements in safety and efficacy characteristics are required. The goals of next-generation  CAR-T designs are to produce universal CAR-T cells from allogeneic donors, increase CAR-T cell  potency, reduce off-target effects, and expand the therapeutic targets beyond liquid cancers.  These new tactics necessitate more intricate CRISPR/Cas9-enabled genetic engineering  techniques, in which the efficiency, safety, and scalability of gene editing are greatly  influenced by the gene delivery strategy selected.^[4]

Genome editing technologies enable scientists to make changes to DNA, leading to changes  in physical traits, like eye color, and disease risk. The first genome editing technologies were  developed in the late 1900s. More recently, a new genome editing tool called CRISPR, invented in 2009, has made it easier than ever to edit DNA. CRISPR is simpler, faster, cheaper,  and more accurate than older genome editing methods. Genome editing is of great interest  in the prevention and treatment of human diseases. Genome editing is used in cells and animal  models in research labs to understand diseases. Scientists are still working to determine  whether this approach is safe and effective for use in people. It is being explored in research  and clinical trials for a wide variety of diseases, including single-gene disorders such as cystic  fibrosis, hemophilia, and sickle cell disease. It also holds promise for the treatment and  prevention of more complex diseases, such as cancer, heart disease, mental illness, and  human immunodeficiency virus (HIV) infection. In the laboratory, scientist edit the genomes  of animals, for example, mice that share about 85 percent of their genes with human! By  changing a single gene or multiple genes in a mouse, scientists can observe how these  changes affect the mouse's health and predict how similar changes in human genomes might  affect human health. The Burgess lab, for example, is studying zebrafish genomes and lab  focuses on 50 zebrafish genes which are similar to the genes that cause human deafness so  that they can better understand the genomic basis of deafness. Using this technique, for the  first time they found CLRN2 is a deafness-causing gene in humans. They established as hearing  loss is probably due to defective protein in the hair cells, where the presence of clarin 2 is  essential for normal organization and maintenance of the mechanosensitive hair bundles. ^[5]

Mutationsin the OTOF gene are a common cause of hereditary hearing loss and the main cause  of auditory neuropathy spectrum disorder (ANSD). As a part of trial Opal Sandy was given  the gene therapy having modified, harmless virus with a working copy of the Otof gene into  the inner ear. Damaged hair cell repaired by the therapy and just a few weeks later, she could  hear loud sounds, such as clapping, in her right ear. A UK girl born deaf can now hear unaided,  after a groundbreaking gene-therapy treatment and since more than half of hearing-loss cases  in children have a genetic cause this therapy is a huge success in medical breakthrough.^[6] 

Gene editing has become more precise and efficient with the development of CRISPR. This  technology allows scientists to better understand diseases and develop new treatments, such  as CAR-T cell therapy for cancer. As of June 2024, the first ever CRISPR-based medicine has  already been approved by the FDA. It is called Casgevy, for treatment of sickle cell disease  and beta thalassemia. CTX001: Developed by CRISPR Therapeutics, it's a CAR-T cell therapy  for multiple myeloma and B-cell lymphoma. It targets the BCMA protein on cancer cells.  ALLOCAR T: Developed by Cellectis, it's another CAR-T therapy for various cancers, including  acute lymphoblastic leukemia (ALL), T-cell lymphoma, and multiple myeloma. Foetal  haemoglobin (HbF) is highly expressed and critical during foetal development, and is then  rapidly suppressed early in life. Reactivation of HbF expression has emerged as an attractive  strategy to treat the symptoms of SCD and beta-thalassemia by compensating for the lack of  functional adult haemoglobin. CTX001 is an autologous, ex vivo cell therapy that is made from  patient’s own haematopoietic stem cells. The stem cells are first collected from a patient’s  blood, and CRISPR-Cas9 is then used to make a small deletion in the B-cell  lymphoma/leukaemia 11A (BCL11A) gene within the genome of these cells. BCL11A is a negative  regulator of HbF expression, thus its disruption increases HbF levels, and this is expected to  lessen the clinical symptoms of beta-thalassemia and SCD.^[7]

Once a far-fetched science fiction idea, genome editing has emerged as a revolutionary force  that is changing the course of biomedical research and treatment. The jewel in this  technology's crown, CRISPR-Cas9, has sped development tremendously and made it possible  to precisely manipulate genetic code with previously unheard-of ease. The ramifications are  enormous, ranging from understanding the complexity of human disease to developing  ground-breaking treatments like CAR-T cells. On the other hand, the problem of off-target  effects highlights the necessity of ongoing improvement and appropriate use. It is impossible  to ignore the ethical implications of genome editing as we approach the dawn of a new era in  medicine. Even if there are a lot of possible advantages, society consequences must be  carefully considered. Still, the trip has only just started. Genome editing has the potential to  eradicate hereditary illnesses, improve human potential, and transform biotechnology and  agriculture with continued research and development. Unquestionably, the future is bright,  but a strong dedication to the welfare of people must guide us down this path with insight,  wisdom, and foresight.

References: 

1. Gaj T, Gersbach CA, Barbas CF 3rd. ZFN, TALEN, and CRISPR/Cas-based methods for genome  engineering. Trends Biotechnol. 2013 Jul;31(7):397-405. doi: 10.1016/j.tibtech.2013.04.004.  Epub 2013 May 9. PMID: 23664777; PMCID: PMC3694601. 

2. Asmamaw M, Zawdie B. Mechanism and Applications of CRISPR/Cas-9-Mediated Genome  Editing. Biologics. 2021 Aug 21;15:353-361. doi: 10.2147/BTT.S326422. PMID: 34456559; PMCID:  PMC8388126. 

3. Zischewski J , Fischer R , Bortesi L. Detection of on-target and off-target mutations  generated by CRISPR/Cas9 and other sequence-specific nucleases. Biotechnology Advances  35 (2017) 95–104. https://doi.org/10.1016/j.biotechadv.2016.12.003. 

4. National Cancer Institute, CAR T Cells: Engineering Patients’ Immune Cells to Treat Their  Cancers. Post date: March 10, 2022; Access date: July 30, 2024. URL:  https://www.cancer.gov/about-cancer/treatment/research/car-t-cells 

5. National Human Genome Research Institute, What is genome editing? Last updated: August  15, 2019; Accessed on: July 30, 2024. URL: https://www.genome.gov/about-genomics/policy  issues/what-is-Genome-Editing. 

6. Andrew Gregory, Medial research UK toddler has hearing restored in world first gene  therapy trial Posted on: 9 May 2024; Accessed on : 30 July 2024. URL:  https://www.theguardian.com/science/article/2024/may/09/uk-toddler-has-hearing-restored in-world-first-gene-therapy-trial

7. Johansen KH. How CRISPR/Cas9 Gene Editing Is Revolutionizing T Cell Research. DNA Cell  Biol. 2022 Jan;41(1):53-57. doi: 10.1089/dna.2021.0579. Epub 2021 Dec 22. PMID: 34939826;  PMCID: PMC8787706.

About the Author:

Shruti  Talashi    

Ms.  Shruti  Talashi  boasts a dual mastery of lab research and writing.  Her doctoral study outcome as M.Phil in biomedical science while studying  breast cancer and an extraordinary masters degrees dissertation work on  exploring role of Gal-lectin in cancer metastasis fuels her extensive  research interests. She has gained few publication in journals. Bridging the  science-public gap is her passion, aided by expertise in diverse techniques.  From oncology to antibiotic/drugs production, she's led and managed complex  projects, even clinical trials. Now, as a freelance Content Coordinator for  Sinoexpo Pharmasource.com, her industry knowledge shines through valuable  insights on cutting-edge topics like GMP, QbD, and biofoundry.