RNA Targeting by Functionally Orthogonal Type VI-A CRISPR-Cas Enzymes

Area of Science:

• Molecular Biology
• Microbiology
• Genetics

Background:

• CRISPR-Cas systems provide prokaryotic immunity against foreign nucleic acids via crRNA-guided nucleases.
• Type VI-A CRISPR-Cas systems utilize the Cas13a protein (formerly C2c2) with dual ribonuclease activities for crRNA maturation and ssRNA degradation.
• The Cas13a protein family exhibits significant variation in protein and crRNA sequence conservation across bacterial phyla.

Purpose of the Study:

• To investigate the functional diversity within the Cas13a enzyme family.
• To determine if Cas13a functional distinctions can be predicted bioinformatically.
• To explore the role of pre-crRNA processing in Cas13a activity and RNA targeting.

Main Methods:

• Phylogenetic analysis of Cas13a protein sequences.
• Biochemical assays to assess ssRNA cleavage specificities.
• Functional characterization of Cas13a subfamilies.
• Investigating the necessity of pre-crRNA processing for Cas13a function.

Main Results:

• The Cas13a enzyme family comprises two distinct functional groups with orthogonal crRNA recognition and different ssRNA cleavage specificities.
• These functional differences were not predictable through bioinformatics, indicating subtle co-evolution.
• Cas13a pre-crRNA processing is not essential for ssRNA cleavage but enhances targeting for internally encoded crRNAs.
• Two Cas13a subfamilies were defined, capable of parallel RNA detection.

Conclusions:

• Cas13a enzymes exhibit functional divergence not discernible by sequence alone.
• The discovery of two distinct Cas13a subfamilies expands the potential for RNA-based diagnostics and understanding bacterial immunity.
• Cas13a's RNA targeting can be modulated by pre-crRNA processing, offering insights into CRISPR-Cas system regulation.