A new study is shedding light on the compact Cas9d RNA-guided nuclease, a smaller variant of the well-known CRISPR-Cas9 systems used for precise genome editing. Researchers reveal the detailed cryo-electron microscopy structures of Cas9d, which consist of only 747 amino acids, providing insights on its innovative DNA targeting mechanism and structural adaptations. The findings may lead to enhanced gene-editing tools with improved efficiency and versatility.
The Cas9d nucleases belong to type II-D CRISPR systems, which are significantly smaller than their type II-A counterparts but nonetheless capable of accurate DNA recognition and cleavage. The structural analysis shows how Cas9d's distinct features—including its ribonucleoprotein architecture—facilitate effective targeting of double-stranded DNA (dsDNA).
Through structural comparison of Cas9d with other nucleases and investigation of its guide RNA (sgRNA), the authors highlighted how this nuclease retains activity even when its guide RNA is truncated by approximately 25%. This characteristic may be pivotal for engineering more compact and efficient gene editing systems.
One key finding from the research is the unique recognition mechanism by which Cas9d interacts with its protospacer adjacent motif (PAM). Unlike other nucleases, which primarily engage PAM through the major groove of the DNA exit, Cas9d involves both major and minor grooves, showcasing its versatile interaction capabilites.
Innovatively, the researchers performed ancestral sequence reconstruction to create an ancestral form of Cas9d, demonstrating significantly higher nuclease activity within mammalian cells as compared to the original Cas9d. This finding suggests promising avenues for therapeutic applications involving genome editing.
The efficiency of the engineered ancestral Cas9d was established through multiple assays, confirming its potential to outperform contemporary counterparts, indicating the underlying evolutionary advantages inherent to the redesigned enzyme.
Understanding how these compact Cas9 systems have adapted their structural properties for efficient DNA interaction stands to inform future research, particularly as scientists strive to streamline CRISPR-Cas technologies for various applications ranging from basic research to clinical therapies.