Structural basis of Cas9 DNA interrogation with a 5' truncated sgRNA

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• CRISPR-Cas9 gene editing efficiency and accuracy are limited by target variability.
• 5' truncated single-guide RNAs (sgRNAs) show promise for reducing off-target effects while maintaining specificity.
• Mechanisms by which truncated sgRNAs enhance specificity and affect Cas9 activation remain unclear.

Purpose of the Study:

• To investigate the structural and biochemical basis of CRISPR-Cas9 activity with 5' truncated sgRNAs.
• To understand how sgRNA truncation impacts Cas9 binding, activation, and specificity.
• To elucidate the role of the non-target strand (NTS) in Cas9 function.

Main Methods:

• Biochemical assays to measure Cas9 activity and binding.
• Cryogenic electron microscopy (cryo-EM) to determine Cas9-sgRNA complex structures.
• Analysis of Cas9-sgRNA interactions and nuclease domain positioning.

Main Results:

• Cryo-EM structures reveal that a 14-nt sgRNA causes steric hindrance of the HNH L1 linker due to the shortened NTS path.
• This steric clash prevents proper positioning of Cas9 nuclease domains, inhibiting cleavage.
• Cleavage inhibition can be overcome by relieving duplex strain or using supercoiled DNA, but plasmid cleavage is ~1000-fold slower than with full-length sgRNAs.

Conclusions:

• The study provides a structural explanation for reduced Cas9 activity with 5' truncated sgRNAs.
• Availability of the PAM-distal NTS is crucial for promoting Cas9 activation and efficient DNA cleavage.
• Findings highlight the importance of sgRNA length and NTS interactions for precise CRISPR-Cas9 genome editing.