Alternatives To CRISPR-Cas9: Nucleases For Next-Gen Therapy

By Rebecca Roberts, Ph.D.

While CRISPR-Cas9 is a widely used gene-editing tool, its limitations have spurred the development of alternative nucleases to improve therapeutic outcomes. Issues like double-stranded breaks (DSBs), off-target effects, and the large size of Cas9, which complicates delivery, have shifted the focus toward other CRISPR systems. Smaller nucleases like SaCas9 and Cas12a are easier to package into adeno-associated viruses (AAVs), making them ideal for in vivo therapies. SaCas9 targets a broader range of genomic regions and fits within AAV size limits, while Cas12a’s staggered cuts improve gene knock-in efficiency.

Other alternatives include Cas3, which can delete large DNA segments, making it effective for gene knockouts, and Cas13, which edits RNA, providing safer options for RNA-based conditions. Additionally, dCas9 and nCas9 avoid creating DSBs, offering safer options for gene regulation and base editing. Cas12e (CasX), a compact nuclease, supports robust editing in mammalian cells.

Advanced variants like hfCas12Max combine high specificity, broad PAM recognition, and multiplexed editing, addressing many of Cas9’s limitations. These nucleases are also being employed in clinical settings, with Cas12a being tested for sickle cell disease and beta-thalassemia, and Cas3 integrated into phage therapies to combat bacterial infections. RNA-targeting Cas13 is being explored for Huntington’s disease treatment.

These alternatives not only broaden the scope of CRISPR-based therapies but also enable safer and more efficient applications. As gene-editing research evolves, newer systems and variants continue to emerge, offering expanded possibilities for clinical and research use.