Intersegmental transfers drive target search in an RNA-targeting CRISPR system

Area of Science:

• Molecular Biology
• Biophysics
• Genetics

Background:

• Sequence-specific RNA-binding proteins (RBPs) must efficiently locate target RNAs.
• Cas13, an RNA-guided CRISPR protein, provides a model for studying RNA target search.
• The mechanism of Cas13's rapid RNA target recognition is unknown.

Purpose of the Study:

• To investigate the mechanism of Cas13's RNA target search.
• To determine how Cas13 achieves rapid target recognition within a critical time constraint.
• To explore the implications of Cas13's search strategy for other RNA-binding proteins.

Main Methods:

• Biophysical modeling
• Activity assays
• Biochemical characterization

Main Results:

• Cas13 utilizes facilitated diffusion to accelerate its search for RNA targets.
• Cas13's search time is inconsistent with 1D sliding, suggesting an alternative mechanism.
• Intersegmental transfers (ITs), involving transient dual binding, are proposed as the primary search mechanism.
• A biophysical model for ITs in RNA was developed and experimentally validated.
• ITs accelerate RNA binding more effectively than DNA binding due to RNA properties.

Conclusions:

• Cas13 employs facilitated diffusion and intersegmental transfers for rapid RNA target recognition.
• Intersegmental transfers represent a potentially widespread mechanism for RNA search by RBPs.
• Understanding Cas13's search strategy illuminates efficient molecular recognition in complex RNA environments.