• CRISPR-Cas9: Advances, Applications, and Future Directions in Biotechnology
  • Bahareh Shahi,1,*
    1. Department of Biology, Research Center for Development of Advanced Technologies, Tehran, Iran


  • Introduction: The rapid evolution of gene-editing technologies over the past two decades has provided increasingly sophisticated platforms for precise manipulation of genetic sequences. The first generation, zinc-finger nucleases (ZFNs), and the second generation, transcription activator-like effector nucleases (TALENs), established the foundation for targeted genome modification. However, the third-generation CRISPR-Cas9 system has emerged as the most widely utilized tool owing to its efficiency, programmability, and cost-effectiveness. Unlike ZFNs and TALENs, which rely on engineered proteins to recognize DNA sequences, CRISPR employs guide RNAs to direct Cas proteins toward specific genomic loci, enabling highly accurate DNA cleavage and repair. Initially identified as an adaptive immune mechanism in bacteria and archaea, CRISPR-Cas9 has been repurposed into a transformative technology with broad applications in agriculture, medicine, environmental sciences, and nanotechnology.
  • Methods: This review integrates evidence from recent studies concerning the applications of CRISPR-Cas9 across diverse domains. Peer-reviewed literature was systematically examined to elucidate both the fundamental mechanisms of Cas9-mediated genome editing and the translational outcomes reported in experimental studies. Emphasis was placed on agricultural innovations in crops and livestock, therapeutic strategies for human genetic disorders, ecological conservation approaches, and the convergence of CRISPR with nanotechnology. Comparative analysis with earlier genome-editing platforms was undertaken, with particular attention to efficiency, specificity, and challenges such as off-target effects, delivery limitations, and immunogenicity.
  • Results: Findings indicate that CRISPR-Cas9 has achieved substantial advances across multiple disciplines. In agriculture, targeted genomic modifications have enhanced disease resistance, grain size, nutritional composition, and breeding efficiency, with studies in rice and wheat demonstrating accelerated improvement cycles relative to conventional breeding methods. In livestock, CRISPR has been employed to improve meat quality, confer disease resistance, and potentially enhance animal welfare, though regulatory and ethical debates remain. In medicine, CRISPR has shown efficacy in correcting pathogenic mutations underlying disorders such as sickle cell disease, retinitis pigmentosa, and familial hypercholesterolemia, highlighting its therapeutic promise. Environmental science has benefited from applications such as engineering resistance in bat populations against white-nose syndrome and exploring genetic approaches for controlling invasive species. Moreover, CRISPR-based biosensing has facilitated the detection of pathogens and pollutants in ecological systems. In nanotechnology, CRISPR integration has enabled more precise delivery of editing components, fine-tuned gene regulation, and epigenetic modifications, thereby expanding its potential in biomedical and research contexts. Collectively, these outcomes underscore the versatility and transformative capacity of CRISPR-Cas9.
  • Conclusion: CRISPR-Cas9 constitutes a paradigm shift in genetic engineering, surpassing earlier tools in precision, adaptability, and applicability. While its integration into agriculture, medicine, environmental science, and nanotechnology has demonstrated considerable promise, unresolved challenges persist, particularly regarding genomic safety, unintended off-target activity, and the ethical implications of germline and ecological modifications. Future directions emphasize the development of high-fidelity Cas9 variants, optimization of guide RNA design, refinement of delivery methodologies, and establishment of robust governance frameworks to ensure ethical and responsible implementation. Ultimately, CRISPR-Cas9 occupies a central position at the forefront of biotechnology, with the potential to redefine both fundamental research and applied sciences, provided its advancement is accompanied by rigorous oversight and societal deliberation.
  • Keywords: CRISPR, CRISPR-Cas9, Gene Editing, Molecular genetic, Genetic engineering