Sharpening the Molecular Scissors: Advances in Gene-Editing Technology

The ability to precisely modify human genes has been made possible by the development of tools such as meganucleases, zinc finger nucleases, TALENs, and CRISPR/Cas. These now make it possible to generate targeted deletions, insertions, gene knock outs, and point variants; to modulate gene expression by targeting transcription factors or epigenetic machineries to DNA; or to target and modify RNA. Endogenous repair mechanisms are used to make the modifications required in DNA; they include non-homologous end joining, homology-directed repair, homology-independent targeted integration, microhomology-mediated end joining, base-excision repair, and mismatch repair. Off-target effects can be monitored using in silico prediction and sequencing and minimized using Cas proteins with higher accuracy, such as high-fidelity Cas9, enhanced-specificity Cas9, and hyperaccurate Cas9. Alternatives to Cas9 have been identified, including Cpf1, Cas12a, Cas12b, and smaller Cas9 orthologs such as CjCas9. Delivery of gene-editing components is performed ex vivo using standard techniques or in vivo using AAV, lipid nanoparticles, or cell-penetrating peptides. Clinical development of gene-editing technology is progressing in several fields, including immunotherapy in cancer treatment, antiviral therapy for HIV infection, and treatment of genetic disorders such as β-thalassemia, sickle cell disease, lysosomal storage disorders, and retinal dystrophy. Here we review these technological advances and the challenges to their clinical implementation.