Scientists have uncovered how Fanzor2’s evolution from its bacterial ancestors could make it a valuable resource for future genomic engineering projects.
The field of biomedicine is evolving rapidly, primarily due to genome engineering technologies like the prokaryotic CRISPR-Cas9 system. Ongoing research is uncovering new genome editing systems in various organisms, which enhances the possibilities for therapeutic applications. Researchers at St. Jude Children’s Research Hospital have explored the evolutionary pathway of Fanzors, which are eukaryotic proteins used for genome editing. Utilizing cryo-electron microscopy (cryo-EM), the scientists have shed light on how Fanzor2 structurally differs from other RNA-guided nucleases, setting the stage for future protein engineering efforts. These findings are published in Nature Structural & Molecular Biology.
CRISPR-Cas9, the genome-editing method that earned the Nobel Prize in Chemistry in 2020, is based on a naturally occurring editing system utilized by bacteria for defense. CRISPR-Cas systems likely stem from transposons, mobile DNA elements. Recently, an extensive and ancient family of transposon-related proteins in bacteria, known as TnpB, has been identified as a functional precursor to various CRISPR-Cas9 and -Cas12 subtypes, serving as an evolutionary link between these processes. The Fanzor family of proteins, which includes Fanzor1 and Fanzor2, are homologous to TnpB, and are found in eukaryotes and certain eukaryotic viruses.
Elizabeth Kellogg, PhD, from the St. Jude Department of Structural Biology, investigated the structure of Fanzor2 to understand how these systems have evolved, providing important insights for future genome engineering technologies.
The potential of Fanzors lies in the link between their structure and function
“After we discovered that TnpBs are also RNA-guided nucleases, similar to CRISPR-Cas9, our interest in their diversity has grown significantly,” Kellogg explained. “They exhibit a wide range of architectures, shapes, and associated RNAs. We are just beginning to discover various biological functions of TnpBs.”
A significant aspect that makes TnpBs and Fanzors particularly interesting is their smaller size compared to Cas9 and Cas12 proteins. This smaller size enhances functionality in genome engineering applications. By analyzing the cryo-EM structures of Fanzor2 in conjunction with its natural RNA guide and DNA target, Kellogg discovered important connections between structure and function in RNA-guided nucleases. This research also indicated that the RNA’s role in shaping Fanzor2’s active site varies from other classes, implying that RNA and protein may have evolved along a different branch than the Cas12 family of CRISPR nucleases.
“The protein is relatively simple, but its structure suggests greater flexibility in how it interacts with its RNAs,” Kellogg noted. “This implies we could potentially further reduce its size, although much more research is required to fully understand this.”
Kellogg is optimistic that this structural data will pave the way for innovative methods in creating the next generation of RNA-guided nucleases. Furthermore, given the diversity within the family, it is apparent that knowledge equates to power. “The structural variety of these complexes is something we currently lack insight into,” she emphasized. “Understanding the functional restrictions that define an RNA-guided nuclease, as well as how to apply these principles in engineering, is crucial. That’s where my interest lies.”
Authors and funding
The primary authors of this study are Richard Schargel from Cornell University, along with Zuhaib Qayyum and Ajay Singh Tanwar from St. Jude. Additionally, Ravi Kalathur from St. Jude contributed to the research.
This study received funding from the National Institutes of Health (R01GM144566), the Pew Biomedical Foundation, the National Science Foundation Graduate Research Fellowship Program, and ALSAC, the fundraising organization for St. Jude.
