Advances in CRISPR-Cas9 Genome Editing: Mechanisms, Variants, Delivery Systems, and Biomedical Applications

Abstract

The CRISPR-Cas9 technology and related programmable nucleases have transformed genome editing by enabling precise and targeted DNA modifications in eukaryotic cells. Compared with earlier tools such as zinc-finger nucleases and TALENs, Cas9 provides higher efficiency and programmability. In practice, optimized systems achieve on-target editing rates exceeding 70–80% in representative mammalian models, while off-target frequencies are often below 1% when using high-fidelity Cas9 variants. A major clinical milestone was reached in 2023 when the U.S. Food and Drug Administration approved the first CRISPR-Cas9–based therapy (exagamglogeneautotemcel, for sickle cell disease and β-thalassemia), demonstrating the translational potential of this platform. Mechanistically, Cas9 functions as an RNA-guided nuclease that requires a protospacer adjacent motif (PAM) for target recognition and cleavage, generating double-strand breaks that are repaired through cellular pathways such as non-homologous end joining or homology-directed repair. Despite its advantages, challenges such as off-target effects, delivery barriers, and immune responses remain. Recent innovations—including base editors, which enable precise single-nucleotide substitutions without double-strand breaks and prime editors, which allow versatile sequence alterations with high accuracy -have further expanded the scope of genome engineering. Collectively, these advances continue to redefine possibilities for biological research and therapeutic applications, providing powerful tools for understanding gene function and developing novel treatments.