Reprogrammable RNA-targeting CRISPR systems evolved from RNA toxin-antitoxins

Area of Science:

• Microbiology
• Molecular Biology
• Evolutionary Biology

Background:

• CRISPR systems are crucial for adaptive immunity in prokaryotes.
• The evolutionary pathways leading to RNA-guided CRISPR systems, particularly Cas13, are not fully understood.
• Understanding CRISPR evolution provides insights into genome defense mechanisms.

Purpose of the Study:

• To identify the evolutionary ancestors of the RNA-targeting CRISPR-Cas13 system.
• To characterize the evolutionary intermediate Cas13e and its relationship with AbiF.
• To elucidate the structural and functional divergence between AbiF and Cas13 systems.

Main Methods:

• Integrated sequence and structure evolutionary tracing.
• Biochemical characterization of Cas13e and AbiF.
• Cryo-electron microscopy (cryo-EM) for structure determination of AbiF.

Main Results:

• Cas13 systems likely evolved from AbiF, a toxin-antitoxin system linked to abortive infection.
• A miniature Cas13, termed Cas13e, was identified as an evolutionary intermediate.
• AbiF functions as a toxin-antitoxin system with an RNA antitoxin, distinct from Cas13's RNA-guided RNA targeting.
• Cryo-EM structure of AbiF revealed structural differences compared to Cas13s.
• Key structural alterations facilitating the evolution from AbiF to CRISPR were mapped.

Conclusions:

• The study provides a clear evolutionary trajectory for RNA-targeting CRISPR-Cas13 systems.
• AbiF represents a plausible precursor to Cas13, highlighting functional and structural transitions.
• The findings deepen our understanding of the evolution of programmable RNA-guided systems.