Structure basis for single-strand nucleic acid targeting by IscB and variants.

Area of Science:

• Molecular Biology
• Structural Biology
• Gene Editing

Background:

• CRISPR-Cas9 technology is derived from the transposon-encoded IscB.
• IscB and related Cas9 enzymes have been engineered into RNA editors by modifying their DNA recognition domains.
• Understanding the mechanism of IscB is crucial for advancing RNA editing tools.

Purpose of the Study:

• To elucidate the structural basis of IscB's interaction with single-stranded nucleic acid (ssNA) targets.
• To understand the mechanism of RNA targeting by IscB, with or without its target-adjacent motif (TAM)-interacting domain (TID).
• To engineer improved IscB variants for enhanced RNA editing efficiency.

Main Methods:

• Four cryo-electron microscopy (cryo-EM) structures of IscB were determined in complex with ssNA targets.
• Structural analysis was performed to understand IscB-ssNA interactions and conformational changes.
• Mutagenesis studies were conducted to alter IscB's ssNA binding and nuclease activity.

Main Results:

• IscB engages ssNA through a conserved mechanism involving initial seed duplex formation and a roadblock by the HNH nuclease.
• Full duplex formation is required to dislodge the HNH roadblock, exposing the nuclease active sites.
• Mutations improving ssNA binding or HNH dislodging enhanced IscB's RNA-targeting efficiency in vitro and in human cells.

Conclusions:

• The structural and mechanistic insights into IscB-ssNA interactions provide a foundation for developing improved RNA editing technologies.
• Engineered IscB variants demonstrate enhanced RNA editing capabilities, paving the way for more precise gene editing applications.
• This research deepens our understanding of RNA-guided endonucleases and their potential in biotechnology.