The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) technology has revolutionized the process of genome editing, making the protein engineering process much more simplified and focused on RNA-directed modification. Initially described as an adaptive immune response in bacteria, CRISPR-Cas9 technology makes possible targeted therapies, plant genome modification, and model organism generation. This review article will describe how Cas9 causes double-stranded DNA breaks, delivery methods of engineered Cas9 and guide RNAs to host cells using viral and non-viral vector systems, as well as new approaches to non-replicating cell genome editing and regulation without DNA breakage. Possible safety limitations such as off-target cleavage, unexpected rearrangement of genome, presence of existing immunity to the vectors and ethical concerns about germline and zygotic editing will be addressed.
Keywords
CRISPR-Cas9, Genome Editing, Single Guide RNA (sgRNA), Gene Therapy, Non-Homologous End Joining (NHEJ), Homology-Directed Repair (HDR).
Introduction
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Genome editing technology allows for accurate modifications of genome DNA sequences in living beings [1, 2]. Prior to the appearance of CRISPR technology, genome editing was achieved using tools such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). However, ZFNs and TALENs technologies were limited by their cost, difficulty of protein design, and technical complexity [2, 12]. The emergence of the CRISPR-Cas9 technology has greatly reduced these obstacles, as it provides for the introduction of a highly modifiable sgRNA that targets the endonuclease to a specific locus in the genome [3, 13].
2. Objectives & Scope
2.1 Objectives
Outline the history of CRISPR-Cas9 development starting from bacterial immune system to current gene-editing platforms [3, 13].
Describe CRISPR-Cas9 applications in human therapeutics, crops modification, and model organism generation (M. musculus, D. melanogaster, A. thaliana) [4, 5, 14, 15].
Analyze delivery systems (e.g., AAV vectors, liposomes), as well as new editing techniques (epigenetic editing, non-proliferating cells' editing) [6, 7, 16].
Discuss issues of ethics, legality, and safety of clinical studies involving human patients and zygote editing [9-11, 17].
2.2 Scope
This review highlights the main experimental results, technical achievements, and clinical achievements described in 52 key literature sources [1-52].
3. Historical Overview & Evolution
Bacterial Origin of CRISPR System: Found as an adaptive prokaryotic immune system protecting bacteria from viruses via cleavage of foreign nucleic acid molecules [3].
Development of Genome Editing Technique: Conversion of the dual-RNA guiding structure into the artificial single guide RNA (sgRNA) cleaving eukaryotic DNA targets [13].
Expanding Model Systems: Efficient use in genetic model organisms such as Drosophila, rodent models, and Arabidopsis thaliana [4, 5, 14, 15].
4. Key Topics & Technological Developments
4.1 Mechanisms of Action & Engineering
Targeted Guide RNA Delivery & DNA Cleavage: Engineered guide RNA directs Cas9 enzyme to desired genomic sites [13]. Cas9 enzyme recognizes PAM sequence and creates a double-strand break (DSB) [3, 13].
Genomic DNA Repair: Double-stranded breaks undergo repair through Non-Homologous End Joining (NHEJ), which results in random insertions of nucleotides at a target site and produces gene knockouts, and through Homology-Directed Repair (HDR), which involves insertion of a sequence via donor DNA [1, 2].
Increasing Efficiency: The introduction of cloning-free CRISPR procedures and rapid construction of endogenous transcriptional reporters [18, 19].
Genetic Modifications: Protocols enabling large fragment deletion and site-specific insertions [8].
Figure 1: CRISPR-Cas9 Working Principle 4.2 Delivery Methods & Vector Systems
Viral-Based: Pre-established AAV delivery system designed for delivery of Cas9 and guide RNAs on a tissue-specific basis [6, 16].
In Zygote: Delivery in one step by injecting the zygote/embryo to produce knock-in and transgenic animals [5, 20].
Figure 2: CRISPR Delivery Protocols
4.3 Therapeutic & Agricultural Applications
Organ Transplant Therapy: Preserving the donor organs in an ex vivo fashion before the transplant surgery [21].
Non-Dividing Cells: Targeted integration methods for gene editing in non-dividing cells without resorting to cell division (homology-independent targeted integration) [7].
Plant Biotechnology: Homozygous mutant plants (crops) developed through the use of Arabidopsis egg cell-specific promoters in one generation [15, 22].
Epigenetic Editing: Fusion of inactive Cas9 protein (dCas9) to transcription factors, thus enabling locus-specific regulation of gene expression without changes in DNA sequence [23, 24].
4.4 Ethical, Safety, & Regulatory Considerations
Genome Integrity: Assessment of off-targets cutting events, chromosomal rearrangements, and large-scale structural deletions [9, 25].
Human Germline Modification: Ethical considerations of human tripronuclei zygote and germline modifications [11, 17, 26].
Regulation of Clinical Trials: Regulatory control over therapy dosages, safety regulations, and intellectual property rights in clinical human trials [10, 27, 28].
5. Future Aspects & Clinical Directions
Epigenetic Editing: Development of dCas9-based gene expression regulators that regulate gene expression without producing DNA double-strand breakage [23, 24].
Improvement of Non-viral Vectors: Modification of lipid nanoparticles (LNPs) to reduce immunogenicity and increase tissue-specificity outside liver cells [29, 30].
Clinical Trials in Humans: Expansion of human trials of treatments for blood diseases, transthyretin amyloidosis, and immunotherapy for cancer [31-33].
International Guidelines: Establishment of international regulatory guidelines for ethics and access to gene therapies [10, 27].
(Figure 3: CRISPR-Cas Applications)
5.1 Extended Clinical & Tool Categories
5.1.1 Human Therapeutics & Clinical Care
Genetic Disorders of Blood: Genetic modification of stem cells outside the body for Sickle Cell Anemia and $\beta$ thalassemia by disabling the enhancer of BCL11A gene [31].
In Vivo Therapy: Genetic editing directly into the liver to treat diseases like transthyretin amyloidosis [32].
Donor Organs: Ex vivo organ perfusion followed by CRISPR targeting to prevent organ rejection [21].
Post-mitotic Tissue Treatment: Targeted integration techniques for post-mitotic tissues [7].
5.1.2 Agriculture & Model Organism Engineering
Plant Tolerance & Productivity: Fast one-generation mutants generated through germline-specific promoter systems [15, 22].
Animal Model Development: Embryonic microinjection in Drosophila and mouse models to establish transgenic models [4, 5, 14, 20].
5.1.3 Advanced Genetic & Molecular Tools
Epigenetic Modulation: Catalytically dead dCas9 conjugated with transcription activators/repressors for transcriptional induction/silencing [23, 24].
Base & Prime Editing: Single-base changes and replacement editing without causing double-strand breaks [34-36].
6. Challenges and Limitations of CRISPR-Cas9
Off-Target Edits: Off-target cutting may occur when there is a mismatch between the sgRNA and genomic DNA sequence [9, 25].
On-Target Structural Changes: Double-strand breaks may result in big deletions, chromosomal changes, or translocations at the target locus [9].
Transport Obstacles: Encapsulation of Cas9 system elements in nontoxic organ-specific vehicles is a significant obstacle; AAVs are burdened by capacity issues, whereas LNPs have difficulty with targeting extrahepatically [6, 29, 30].
Reaction of the Immune System: The prior presence of antibodies and T cells to Cas9 proteins from S. aureus and S. pyogenes can be detrimental for treatment clearance and immune reactions [37, 38].
Inefficiency in Post-Mitotic Cell Types: Repair of DSB through HDR takes place mostly in dividing cells, hindering the replacement of genes in non-dividing post-mitotic tissues such as neurons [7].
Ethical and Accessibility Issues: Financial costs restrict global availability, while the germline and embryonic edition have long-term ethical consequences [10, 11, 17, 26, 27].
7. Case Studies
7.1 Sickle Cell Disease & β
-Thalassemia (Ex Vivo Stem Cell Therapy)
Approach for Treatment: Ex vivo gene editing of the patient’s own CD34+ hematopoietic stem and progenitor cells (HSPCs) [31].
Molecular Mechanism: CRISPR-Cas9 is used to target the erythroid-specific enhancer domain of BCL11A [31]. The disruption of this enhancer prevents the transcription of BCL11A and activates the production of fetal hemoglobin HbF (α2γ2) to prevent sickling of the red blood cells [31].
Clinical Evidence: The patients showed increased levels of HbF, absence of vaso-occlusive crises, and independence of blood transfusion [31].
(Figure 4: Pathway for Ex Vivo Editing of HSPCs)
7.2 Cancer Immunotherapy (Engineered CAR-T Cells)
Approach for Treatment: T-cells from the patient or donors are first subjected to multiple rounds of CRISPR-Cas9 editing ex vivo prior to re-infusion.
Molecular Mechanism: Inactivation of the PDCD1 gene, which is responsible for producing PD-1, will hinder the development of exhaustion in T-cells due to the tumor [39]. Knockout of both endogenous T-cell receptor genes (TRAC/TRBC) and HLA genes allows for off-the-shelf allogenic CAR-T cells to be used without GvHD [33, 39].
Clinical Evidence: Gene editing for multiple genes has led to improvements in CAR-T persistence and antitumor effectiveness [33, 39].
(Insert Figure 5: Workflow of Engineered CAR-T Cells)
8. Declarations
Conflict of Interest: Authors report no conflict of interest concerning the publication of this manuscript.
Financial Disclosure: This manuscript did not receive any specific funding.
9. CONCLUSION
The CRISPR-Cas9 technology has marked a breakthrough in molecular biology and experimental medicine [1, 2]. The problems that need solving are those of off-target cleavage, delivery of targets, immunity, and bioethical issues [6, 9, 10, 37]. Improvements in base editing, prime editing, dCas9-based epigenetic control, and non-viral delivery systems will help increase the scope of use for gene-editing therapies [23, 29, 34, 35].
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Jiya Goswami
Corresponding author
Pioneer Pharmacy College, At & Post: Sayajipura, Ajwa-Nimeta Road, Vadodara, Gujarat, India.
Vani Varia
Co-author
Pioneer Pharmacy College, At & Post: Sayajipura, Ajwa-Nimeta Road, Vadodara, Gujarat, India.
Yagnesh Modi
Co-author
Assistant Professor, Pioneer Pharmacy College, At & Post: Sayajipura, Ajwa-Nimeta Road, Vadodara, Gujarat, India.
Satyajit Sahoo
Co-author
Associate Professor, Pioneer Pharmacy College, At & Post: Sayajipura, Ajwa-Nimeta Road, Vadodara, Gujarat, India.
D.B. Meshram
Co-author
Principal, Pioneer Pharmacy College, At & Post: Sayajipura, Ajwa-Nimeta Road, Vadodara, Gujarat, India
10.5281/zenodo.22967571