The Optimized Micro IscB-ωRNA System Has Great Application Potential in Gene Editing Therapy

Recently, researchers published a research paper titled "Engineered IscB-ωRNA system with improved base editing efficiency for disease correction via single AAV delivery in mice" in the journal Cell Reports. The study successfully engineered the IscB-ωRNA system, a transposon-related CRISPR ancestral system. The gene knockout and base editing efficiency of IscB-ωRNA were improved, and the feasibility of the optimized IscB-ωRNA system for gene editing therapy was verified in a mouse metabolic disease model. The optimized micro-IscB-ωRNA system can be delivered via a single adeno-associated virus (AAV) vector, and has great application potential in gene editing therapy.

In this latest study, the research team focused on the transposon-related RNA guide nuclease IscB, a CRISPR ancestor protein that is much smaller than the traditional Cas9 and Cas12 proteins. IscB's miniaturized protein size allows it to be more easily packaged into a single AAV vector, making it suitable for in vivo delivery. However, the gene editing efficiency of natural IscB protein in eukaryotic cells is low, limiting its widespread application.

Through structural analysis of the IscB-ωRNA protein nucleic acid complex, Xu Chunlong's team conducted multiple rounds of systematic engineering modifications of IscB-associated ωRNA and successfully discovered a new variant of ωRNA*-v2. This improved ωRNA significantly improved the gene knockout and base editing efficiency of IscB. In mouse embryo experiments, the Tyr gene and DMD gene were successfully edited using the IscB-ωRNA system, demonstrating the great potential of this system in in vivo gene editing.

Figure 1. The top shows the RNA structure of the engineered ωRNA*-v2 variant; the bottom shows the ωRNA*-v2-guided IscB gene knockout and base editing tools and their applications in disease mouse models. (Guo R, et al., 2024)

In addition, the research team also conducted an in-depth analysis of the off-target effects of the system. Through genome-wide PEM-seq analysis, the results showed that the modified IscB-ωRNA system did not cause significant off-target effects during the gene editing process. At the same time, the research team also conducted R-loop and RNA-seq analysis to further evaluate the gene editing specificity of miCBE and miABE. The study found that compared with traditional SpCas9-derived gene editors, the IscB-ωRNA system caused a lower number of non-target edits and showed better specificity.

Finally, the research team tested the therapeutic effect of this tool in a mouse model of inherited metabolic diseases. Through a single AAV delivery of IscB-ωRNA* gene knockout and single-base editing system, they successfully repaired the single-base mutation in the Fah gene, reversed the death phenotype of the model mice and improved the health of the diseased mice. This discovery not only proves the effectiveness of the engineered IscB-ωRNA system in in vivo gene editing, but also demonstrates its potential in disease gene editing therapy.

Reference

Guo R, et al. Engineered IscB-ωRNA system with improved base editing efficiency for disease correction via single AAV delivery in mice. Cell Reports, 2024, 43(11).