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A New Era in Genome Editing: Insights from St. Jude Children’s Research Hospital

The field of biomedicine is experiencing a significant transformation, largely facilitated by innovative genome engineering technologies such as the prokaryotic CRISPR-Cas9 system. Researchers continue to discover new genome editing mechanisms across various organisms, expanding the possibilities for therapeutic interventions. A recent study from St. Jude Children’s Research Hospital has focused on Fanzors, a family of eukaryotic proteins involved in genome editing. Utilizing cryo-electron microscopy (cryo-EM), the team revealed structural differences of Fanzor2 in comparison to other RNA-guided nucleases, setting the stage for advancements in protein engineering. These findings have been published in Nature Structural & Molecular Biology.

CRISPR-Cas9, which earned the Nobel Prize in Chemistry for its groundbreaking utility, was adapted from a natural genome-editing system used by bacteria for defense against viruses. Recent findings suggest that CRISPR-Cas systems may have evolved from transposons—DNA segments capable of relocating within the genome. Notably, the discovery of TnpB, a large protein family associated with transposons in bacteria, has illustrated its role as a precursor to multiple CRISPR-Cas subtypes, effectively bridging an evolutionary gap. The Fanzor family, which includes Fanzor1 and Fanzor2, functions as homologs of TnpB in eukaryotes and their viruses.

Dr. Elizabeth Kellogg from St. Jude’s Department of Structural Biology has been investigating Fanzor2’s structure to monitor the evolution of these systems, providing valuable insights for future genome engineering techniques.

Exploring the Structure-Function Relationship of Fanzors

The potential of Fanzors is significantly tied to their unique structure-function relationships.

“The discovery that TnpBs function as RNA-guided nucleases similar to CRISPR-Cas9 has reignited our interest in their diverse characteristics,” Kellogg noted. “These proteins exhibit a remarkable variety in architecture, shapes, and associated RNAs. We are just beginning to uncover their numerous biological roles.”

A noteworthy advantage of TnpBs and Fanzors is their relatively small size compared to Cas9 and Cas12 proteins. This characteristic is particularly beneficial for genome engineering, as smaller proteins can enhance functional versatility. In Kellogg’s research, cryo-EM images depicting Fanzor2 in conjunction with its native RNA guide and DNA target helped outline the intricate relationship between a nuclease’s structure and its function. The analysis also indicated that the RNA associated with Fanzor2 plays a distinct role in constituting the active site, which diverges from the characteristics observed in the Cas12 family of CRISPR nucleases.

“Although the protein appears minimal, our findings suggest much greater flexibility in how these proteins function with their associated RNAs,” Kellogg observed. “This points towards the possibility of further size reduction, but there is still much work required to comprehend this process fully.”

Kellogg expresses hope that this foundational understanding will pave the way for creating the next generation of RNA-guided nucleases. Given the extensive diversity within the Fanzor family, knowledge derived from this research is poised to empower future innovations. “We still have scant understanding of the structural diversity within these complexes,” she emphasized. “This knowledge is pivotal, not only in grasping the functional constraints that categorize an RNA-guided nuclease but also in leveraging these insights for engineering applications. That is where my primary interest lies.”

Study Contributors and Funding

The study’s lead authors include Richard Schargel from Cornell University, along with Zuhaib Qayyum and Ajay Singh Tanwar from St. Jude. Ravi Kalathur from St. Jude also contributed to the research.

This research received financial support from a variety of sources, including the National Institutes of Health (R01GM144566), the Pew Biomedical Foundation, the National Science Foundation Graduate Research Fellowship Program, and ALSAC, the fundraising body of St. Jude.

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