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Pradip Bhattacharjee1 & Dr. Sanjukta Manna2 Student of 4th Semester, B.Sc. Zoology, Maulana Azad College 2 Assistant Professor, Dept. of Zoology, Maulana Azad College
Abstract: Genome editing technology holds great promise for genome manipulation and gene therapy. A novel gene-editing technique, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9, has recently emerged as an efficient tool for inducing targeted genetic modifications. The revolutionary feature of this technology is that one nuclease domain of Cas9 cleaves the target strand of DNA and another nuclease domain cleaves the non-target strand and this ability to modify selectively specific genes provides a powerful tool for characterizing gene functions, performing gene therapy, correcting specific genetic mutations and eradicating diseases. The application of this new technology to stem cell research allows disease models to be developed to explore new therapeutic tools. Stem cells are defined by their long-term self-renewal and their ability to differentiate into specialized progeny. Cell therapy is the administration of live cells to a patient with the aim of repairing or replacing damaged cells or tissues. Thus genome edited stem cells with this technology has created a new perspective in stem cell therapy and is becoming the point of focus day by day for the advent of new therapeutic tools for several degenerative diseases.
Genome Editing is widely used in biological research, especially in stem cell research on human diseases. Genome editing technologies based on common engineered nuclease-based platforms, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindrome repeats (CRISPR) in combination with Cas9 RNA-guided endonucleases, utilize the endogenous DNA repair machinery to easily target almost any genomic location (fig.1). History of CRISPR/Cas: In 1987, CRISPR was identified in Escherichia coli as an adaptive immune system against plasmids, viral DNA or RNA. All CRISPR loci acquire spacers from foreign DNA elements. So far, numerous CRISPR systems have been identified. The most commonly used CRISPR/Cas9 system is the type II system, and its biology, mechanism, and applications have been well-documented.
Fig.1- Genome Editing Techniques
Strategies of Genome-Editing with CRISPR/Cas: The CRISPR/Cas9 system consists of two components, a Cas9 nuclease and a single guide RNA. The latter consists of sequence-specific targeting crRNA and tracrRNA, which bind to Cas9 together with the CRISPR RNA and guide the nucleases for sequence-specific cleavage of complementary sequences. There are three different CRISPR/Cas9-based genome editing strategies, 1) plasmid-based CRISPR/Cas9 system, 2) Cas9/mRNA and sgRNA system, and 3) ribonucleoprotein (RNP) complex-based Cas9 and sgRNA system. Each strategy has its advantages and shortcomings . Among the strategies, the RNP system is most commonly used for clinical purposes as it is associated with fewer off-target effects. The mechanism of action is given in the (fig.2).
Stem Cells are undifferentiated cells that can turn into specific cells, as the body needs them. Stem cells originate from two main sourcesadult body tissues and embryos. There are different types of stem cells based on their potency, such as totipotent, pluripotent, multipotent stem cells. Genome-Edited Stem Cells for Therapeutic Application: Stem cells are an optimal platform for genome editing technologies owing to their self-renewal capability and ability to secrete endogenous proteins. In ex vivo gene editing-based stem cell therapy, defective genes are isolated from patient-derived cells, repaired in vitro using gene editing technologies, and then, transplanted back into the patient. In vivo genome editing in endogenous stem cells is performed using either viral vectors or a mixture of viral vectors and lipid nanoparticles (fig.3).
Fig.3- Action of CRISPR on Genomic DNA and Its Control
Treatment with Genome-Edited Stem Cells: Several degenerative genetic diseases can be treated with this emerging technology. The below mentioned list is some of them. Stem Cell Type
Diseases
Hematopoietic stem and progenitor cells
Ex vivo: X-linked chronic granuloma X-linked SCID Sickle cell disease Beta thalassemia
Neural stem cells
Brain tumor
Skin stem cells
In vivo: Recessive dystrophic epidermolysis bullosa Duchenne muscular dystrophy
Cardiomyocytes and muscle stem cells Advantages
Fig.2- Mechanism of Action of CRISPR/cas9
Target design simplicity; Multiplexed target recognition; Efficiency for editing; Screening DNA noncoding parts.
Disadvantages Limited PAM sequences; Off-site effects; Mosaicism; Costly and time-consuming.
Conclusion: Genome-edited stem cell therapy has advantages over traditional stem cell therapies such as, functional improvement, mutation correction, homing and survival improvement, human leukocyte antigen matching, and highly efficient treatment. However, the challenges associated with the strategies for precise ex vivo and in vivo editing and the delivery of gene-editing endonucleases must be addressed before genome editing is approved as a therapeutic tool and becomes incorporated into clinical practices in near future. Acknowledgement: I want to sincerely thank Dr. Subir Chandra Dasgupta, Head of the Department of Zoology, Maulana Azad College for helping and guiding us in this work, and also to the organizers for giving us such an wonderful opportunity. References: 1. S., M., Byrne, P., Mali, G., M., Church. Genome editing in human stem cells. Methods Enzymol. 546, 119–138 (2014). 2. F., A., Ran et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281–2308 (2013). 3. A., M. Moreno, P., Mali. Therapeutic genome engineering via CRISPR-Cas systems. Wiley Interdiscip. Rev. Syst. Biol. Med. 9, e1380 (2017). 4. K., Takahashi, K., Okita, M., Nakagawa, S., Yamanaka. Induction of pluripotent stem cells from fibroblast cultures. Nat. Protoc. 2, 3081–3089 (2007).