The structure of a eukaryotic CRISPR-Cas homolog, Fanzor2, shows promise for gene editing

Scientists at St. Jude Children’s Research Hospital have revealed how Fanzor2’s divergence from bacterial ancestors could make it a useful tool for future genome engineering projects.

A revolution in biomedicine is currently underway, driven by the application of genome engineering tools such as prokaryotic CRISPR-Cas9. Novel genome editing systems continue to be identified in different organisms, adding to the potential toolbox for various therapeutic applications.

Scientists at St. Jude Children’s Research Hospital studied the evolutionary path of Fanzors, eukaryotic genome-editing proteins.

Using cryo-electron microscopy (cryo-EM), researchers provided insight into the structural divergence of Fanzor2 from other RNA-guided nucleases, providing a framework for future engineering projects proteins. The results were published in Nature Structural and molecular biology.

CRISPR-Cas9, the genome editing approach that won the Nobel Prize in Chemistry in 2020, was adapted from a natural genome editing system that bacteria use as a defense mechanism. CRISPR-Cas systems can arise from transposons, DNA elements that move from one genomic location to another.

Recently, a large and ancient family of transposon-associated proteins found in bacteria, called TnpB, was found to be a functional predecessor of several CRISPR-Cas9 and -Cas12 subtypes, providing an evolutionary bridge between the two processes. The Fanzor family of proteins, consisting of Fanzor1 and Fanzor2, are homologs of TnpB found in eukaryotes and eukaryotic viruses.

Elizabeth Kellogg, Ph.D., Department of Structural Biology at St. Jude, studied the structure of Fanzor2 to trace the evolution of these systems, providing key insights to inform future approaches to plant engineering technology. genome.

Fanzor’s potential lies in its structure-function relationship

“Since it was discovered that TnpBs are also RNA-guided nucleases, much like CRISPR-Cas9, we have been very interested in their diversity,” Kellogg explained. “They have a lot of variety in terms of architecture, shapes and the RNAs associated with them. We are just now discovering all sorts of biological roles for TnpBs.”

A key factor that makes TnpB and Fanzors so interesting is their relative size: they are significantly smaller than their Cas9 and Cas12 relationships. In terms of genome engineering, minimizing the size of the protein provides more functionality.

Using cryo-EM structures of Fanzor2 combined with its native RNA guide and DNA target, Kellogg reconstructed the relationship between structure and function in RNA-guided nucleases. The work also revealed that the role of RNA in patterning the active site of Fanzor2 differs from that of other classes, suggesting that RNA and protein co-evolved on a distinct evolutionary branch of the Cas12 family. CRISPR nucleases.

“The protein was pretty minimal, but the structure suggests there’s a lot more malleability in terms of how it works with their RNAs,” Kellogg said. “This suggests that we could reduce its size further, but there is still a lot of work to be done to understand this.”

Kellogg hopes this structure will be the launching pad for new approaches to designing the next generation of RNA-guided nucleases. Additionally, given the diversity of the family, it is clear that with knowledge comes power.

“The structural diversity of these complexes is simply something we have no understanding of,” she stressed. “That’s where I think it’s important, not only to understand the functional constraints that make something an RNA-guided nuclease, but also to understand those principles and exploit them in engineering. That’s what which interests me.”