A structural accessibility principle for LbuCas13a activation by noncontiguous DNA

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• CRISPR-Cas13a enzymes are primarily known as RNA-guided RNA endonucleases.
• Leptotrichia buccalis Cas13a (LbuCas13a) exhibits DNA-binding and activation capabilities.
• Understanding DNA-mediated activation, especially with noncontiguous DNA, is crucial for expanding CRISPR applications.

Purpose of the Study:

• To define the structural accessibility principle for LbuCas13a activation by noncontiguous DNA.
• To investigate the impact of DNA overhang positioning on LbuCas13a activity.
• To explore LbuCas13a's potential for sensitive biomarker detection.

Main Methods:

• Structural analysis of LbuCas13a-DNA interactions.
• Molecular modeling and molecular dynamics simulations.
• Development of CRISPR-based detection assays (ACROSS).

Main Results:

• LbuCas13a activation by noncontiguous DNA is dictated by accessible protein-DNA interfaces created by overhang positioning.
• Outer overhangs near the crRNA repeat-adjacent side enhance trans-cleavage activity.
• Inner overhangs hinder activation due to steric clashes.
• Noncontiguous DNA broadens LbuCas13a's discrimination window, enabling accurate IDH1 R132H detection in glioma tissues.
• The ACROSS system allows sensitive detection of APE1 and stable signaling in various sample types.

Conclusions:

• The structural accessibility principle governs LbuCas13a activation by noncontiguous DNA.
• LbuCas13a's DNA-mediated activation can be precisely controlled by DNA structure.
• CRISPR-LbuCas13a systems, like ACROSS, offer sensitive and stable detection platforms for clinical biomarkers.