The ability to precisely edit RNA offers a transformative approach to medicine, enabling transient, reversible interventions for genetic diseases without the risks of permanent DNA alteration. However, the field has been hampered by a critical bottleneck: the primary tool, CRISPR-Cas13, suffers from "collateral cleavage" activity, an indiscriminate shredding of bystander RNA molecules that leads to significant cellular toxicity. This has largely stalled its therapeutic translation.
Now, a landmark study in Cell by Xu et al. introduces an elegant and powerful solution, not by discovering a new system, but by repurposing ancient ones [1]. By applying a simple yet profound engineering principle, the researchers have converted the DNA-targeting enzymes IscB and Cas9 into a new class of highly efficient, specific, and—most importantly—cytotoxicity-free RNA editors.
The journey of gene editing has been dominated by DNA-targeting CRISPR-Cas systems, most famously Cas9. The discovery that these bacterial immune systems could be programmed to edit genes in human cells revolutionized biology. Soon after, attention turned to their RNA-targeting counterparts, the Cas13 family, which promised a safer, non-permanent therapeutic strategy [4].
However, the initial excitement for Cas13 was tempered by the discovery of its collateral activity. Upon binding its target RNA, Cas13 becomes a hyperactive, non-specific ribonuclease, degrading RNAs throughout the cell and causing widespread toxicity, a major barrier for clinical use [5]. The field was at an impasse, in need of a tool with the programmability of Cas13 but without its dangerous side effects. The answer, it turns out, was hiding in the evolutionary history of Cas9 itself. In 2021, researchers identified IscB, a compact RNA-guided DNA-cutting enzyme from the IS200/IS605 transposon family, as a likely evolutionary ancestor of Cas9 [2]. Subsequent structural studies revealed how IscB and its guide RNA (ωRNA) function, providing a blueprint for its engineering [3, 6].
The work by Xu et al. began with a key observation: IscB, while primarily a DNA-targeting enzyme, possesses an intrinsic, albeit weaker, affinity for single-stranded RNA. The team hypothesized that its preference for DNA was enforced by a specific protein domain responsible for recognizing a "target-adjacent motif" (TAM) on the DNA strand. Their innovative idea was brilliantly simple: what if they just removed it?
By deleting this small TAM-interacting domain (TID), they created a new protein they termed R-IscB. This single modification effectively "reprogrammed" the enzyme, switching off its DNA-binding capability and unleashing its now-dominant RNA-targeting function. The result is an ultracompact RNA editor that is not only as efficient as Cas13 but completely lacks its toxic collateral activity.
The researchers rigorously validated R-IscB's versatility across multiple therapeutic applications:
Perhaps the most significant finding was the generalizability of this engineering principle. The team applied the same logic to Cas9, the workhorse of DNA editing. By deleting the corresponding PAM-interacting domain (PID) in four different Cas9 orthologs, they successfully converted all of them into efficient, RNA-guided RNA-targeting tools, proving that this "domain deletion" strategy is a universal blueprint for repurposing a vast family of nucleases.
This study represents more than just the creation of a new tool; it establishes a new paradigm for engineering biological systems. It demonstrates that by understanding the evolutionary and structural logic of a protein, we can unlock entirely new functions through minimalist, targeted modifications. The R-IscB and Cas9-ΔPID systems are not only safer but also significantly smaller than Cas13, a critical advantage for in vivo delivery using vehicles like adeno-associated viruses (AAVs).
The implications are profound. We now have a generalizable strategy to mine the immense natural diversity of CRISPR-like systems and convert them into bespoke RNA-targeting tools for therapy, diagnostics, and research. The next frontier will involve scaling this discovery process. Engineering the next generation of editors will require massive libraries of designs to be built and tested. Platforms that enable the autonomous screening of thousands of genetic constructs, such as Ailurus vec, could dramatically accelerate the optimization of these novel RNA editors by linking function to survival in a single culture.
By looking to the evolutionary past of our most powerful gene editors, Xu et al. have charted a clear path toward a safer and more versatile future for RNA therapeutics. This work not only solves a critical problem but also provides a powerful new blueprint for innovation in the age of synthetic biology.
Ailurus Bio is a pioneering company building biological programs, genetic instructions that act as living software to orchestrate biology. We develop foundational DNAs and libraries, transforming lab-grown cells into living instruments that streamline complex research and production workflows. We empower scientists and developers worldwide with these bioprograms, accelerating discovery and diverse applications. Our mission is to make biology the truly general-purpose technology, as programmable and accessible as modern computers, by constructing a biocomputer architecture for all.
