C2c1-sgRNA Complex Structure Reveals RNA-Guided DNA Cleavage Mechanism

Area of Science:

• Molecular Biology
• Structural Biology
• Genetics

Background:

• CRISPR-Cas systems are microbial adaptive immune mechanisms utilized for genome editing.
• Type V-B CRISPR-Cas systems, like C2c1, are increasingly explored for biotechnological applications.
• Understanding the structural basis of C2c1-guide RNA interaction is crucial for optimizing its use.

Purpose of the Study:

• To determine the crystal structure of Alicyclobacillus acidoterrestris C2c1 (AacC2c1) in complex with a single-molecule guide RNA (sgRNA).
• To elucidate the mechanism of sgRNA binding and its implications for C2c1 function.
• To assess the specificity of the AacC2c1-sgRNA system for genome editing applications.

Main Methods:

• X-ray crystallography was employed to obtain the high-resolution structure of the AacC2c1-sgRNA complex.
• Biochemical assays were performed to evaluate the system's sensitivity to guide RNA-target DNA mismatches.
• Structural analysis focused on the sgRNA scaffold, its interaction with C2c1 domains, and PAM recognition.

Main Results:

• The crystal structure revealed a bi-lobed AacC2c1 architecture (REC and NUC lobes) with a tetra-helical sgRNA scaffold, differing from prior predictions.
• The crRNA component of sgRNA binds in the central channel, while the tracrRNA occupies an external groove.
• Despite lacking a canonical PAM-interacting domain, AacC2c1 demonstrated specific PAM duplex binding and high sensitivity to single-nucleotide mismatches, indicating reduced off-target cleavage.

Conclusions:

• The sgRNA assembly and binding mechanism in AacC2c1 are distinct from those observed in Cas9 and Cpf1.
• The high specificity of the AacC2c1-sgRNA system makes it a promising tool for precise genome editing.
• Structural insights into AacC2c1 provide a foundation for further engineering of CRISPR-based technologies.