Genome Editing Beyond Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR): Comparative Insights into Alternative Tools

Abstract

Genome editing has been revolutionized by the introduction of CRISPR–Cas systems, which, in a very short time, became the main tools in both research and clinical pipelines. Nevertheless, CRISPR-based methods have their limitations. Unintended targeting, protospacer adjacent motif (PAM) requirements, delivery difficulties, and possible immunogenicity are still the main challenges for a widely safe application. These limitations have led to the revival of older genome-editing technologies such as meganucleases, zinc-finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs), as well as to newer inventions like base editors and prime editors. This review covers alternative genome-editing platforms that go beyond the use of classical CRISPR nucleases. The discussion covers molecular mechanisms, applications, and performance compared to CRISPR systems. Delivery strategies, such as viral, non-viral, and mRNA/RNP approaches, are examined along with their suitability for ex vivo and in vivo applications. Clinical case studies, including CCR5 editing for HIV, hemoglobinopathies, and in vivo therapies for liver and eye disorders, demonstrate real-world applications. Ethical and regulatory issues are also addressed, particularly regarding germline versus somatic editing, patient consent, long-term monitoring, and international policies. Analysis reveals that no single approach is best in all cases, and the choice of tools depends largely on the biological question or therapeutic goal. Next steps for these technologies include combining and refining methods and further developing the genome-editing toolkit to ensure safe and efficient clinical translation.