A revolution in biomedicine is currently underway, driven by the application of genome engineering tools such as the prokaryotic CRISPR-Cas9. New 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 journey of Fanzors, eukaryotic genome-editing proteins. Using cryo-electron microscopy (cryo-EM), the researchers provided insights into the structural divergence of Fanzor2 from other RNA-guided nucleases, proposing a framework for future protein engineering endeavors. The findings were published today in Nature Structural & Molecular Biology.

CRISPR-Cas9, the genome-editing approach that won the Nobel Prize in Chemistry in 2020, was adapted from a naturally occurring genome editing system bacteria use as a defense mechanism. CRISPR-Cas systems may have originated from transposons, DNA elements that move from one genomic location to another. Recently, a large and ancient transposon-associated protein family found in bacteria, called TnpB, was discovered to be a functional predecessor to multiple CRISPR-Cas9 and -Cas12 subtypes, providing an evolutionary bridge between the two processes. The Fanzor protein family, comprised of Fanzor1 and Fanzor2, are homologs of TnpB found in eukaryotes and eukaryotic viruses.

Elizabeth Kellogg, PhD, St. Jude Department of Structural Biology, studied the structure of Fanzor2 to chart how these systems have evolved, offering key insights to inform future approaches to genome engineering technology.

Fanzor potential lies in its structure-function relationship

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

One key factor that makes TnpBs and Fanzors so exciting is their relative size -- they are significantly smaller than their Cas9 and Cas12 relations. In terms of genome engineering, minimizing the size of the protein offers more functionality. Through cryo-EM structures of Fanzor2 associating with its native RNA guide and DNA target, Kellogg pieced together the relationship between structure and function in RNA-guided nucleases. The work also revealed that RNA's role in helping to structure the active site of Fanzor2 differs from other classes, suggesting the RNA and protein co-evolved on a separate evolutionary branch from the Cas12 family of CRISPR nucleases.

"The protein was pretty minimal, but the structure suggests there's way more malleability in terms of how they function with their RNAs," Kellogg said. "It hints that we could reduce its size further, but there's a lot more to be done to understand that."

Kellogg hopes this structure will be the launchpad for novel approaches to engineering the next generation of RNA-guided nucleases. Moreover, considering the diversity of the family, it is clear that with knowledge comes power. "The structural diversity of these complexes is just something that we have no understanding of at all," she emphasized. "That's where I think it's important, not only for understanding the functional constraints that make something an RNA-guided nuclease, but also how you understand those principles and harness them in engineering. That's what I'm interested in."

Authors and funding

The study's first authors are Richard Schargel, Cornell University; and Zuhaib Qayyum and Ajay Singh Tanwar, St. Jude. The study's other author is Ravi Kalathur, St. Jude.

The study was supported by grants from the National Institutes of Health (R01GM144566), Pew Biomedical Foundation, the National Science Foundation Graduate Research Fellowship Program and ALSAC, the fundraising and awareness organization of St. Jude.