A recent study published in Nature Communications unveils groundbreaking insights about Cas9d, the smallest member of the CRISPR-Cas9 enzyme family, by detailing its structure and mechanisms involved in DNA cleavage. Researchers from Deltaproteobacteria successfully characterized the Cas9d protein and its sgRNA (single-guide RNA) through cryo-electron microscopy, providing structural models for both target-free and target-bound states.
CRISPR-Cas9 systems are known for their ability to edit genes by targeting specific DNA sequences. The dual-RNA guided system typically utilizes the Cas9 enzyme, CRISPR RNA (crRNA), and trans-activator crRNA (tracrRNA) to facilitate the precise cleavage of invasive DNA, such as from viruses or plasmids. The addition of the sgRNA simplifies this process by linking the crRNA and tracrRNA, streamlining genomic editing applications.
The team found out how Cas9d employs a compact architecture for target recognition and cleavage; it employs 747 amino acids and utilizes only 135 nucleotides for its sgRNA scaffold. A key aspect of their study demonstrated the requirement of at least 17 base pairs of stable guide-target heteroduplex formation for effective nuclease activity, affirming the Cas9d system's potential for targeted genomic modifications.
Upon examining the bonding interactions within the Cas9d system, researchers identified its unique PAM (protospacer-adjacent motif) recognition mechanism which is reliant on its WED (wedge) and PI (PAM interacting) domains. The study highlighted how specific amino acids form hydrogen bonds with the PAM, primarily recognizing the NGG sequence downstream of the target DNA, which is aligned with typical CRISPR processes.
Further investigations revealed the structural intricacies between the Cas9d scaffold and its REC (target recognition) domain, establishing them as integral components of what the study terms the “RNA-coordinated target Engagement Module” (REM). This hybrid functional module was found to undergo conformational changes upon target binding, which are pivotal for accurately cleaving target DNA and maintaining low tolerance for mismatches.
To address the limitations of size imposed by traditional CRISPR systems, the researchers implemented engineered modifications to the Cas9d and its sgRNA. By optimizing both components, they successfully produced a compact system without sacrificing nuclease activity. The compact expression cassette of Cas9d is less than 3.4 kb, making it particularly relevant for applications using adeno-associated virus (AAV) delivery, which is limited to 4.7 kb.
Experimental results demonstrated Cas9d's efficacy, exhibiting double-stranded (ds) DNA cleavage activity, plasmid clearance within E. coli, and base editing capabilities within human cells at up to 22% efficiency. Notably, biochemical assays underscored its more stringent mismatch tolerance compared to other Cas9 proteins, such as SpyCas9, solidifying Cas9d's position as a high-fidelity genome editing tool with precise target engagement.
This study points to significant future directions for Cas9d-based applications, particularly as engineers continue to explore variations to broaden the system's functionality and fidelity. The findings contribute to the growing body of literature aimed at enhancing genome-editing methods using compact CRISPR systems.
The comprehensive structural and mechanistic insights obtained from the study serve as foundational knowledge for enhancing genomic tools with improved precision and reduced off-target effects, marking Cas9d as one of the most exciting developments within genetic engineering to date.