tRNA anticodon cleavage by target-activated CRISPR-Cas13a effector

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Type VI CRISPR-Cas systems are RNA-targeting systems that provide adaptive immunity in bacteria.
• Cas13 effectors, upon binding target RNA, exhibit non-specific RNAse activity, leading to collateral cleavage.
• This collateral activity is proposed to induce bacterial dormancy and protect populations from phage infection.

Purpose of the Study:

• To investigate the specific RNA targets of collateral cleavage by Leptotrichia shahii Cas13a (LshCas13a) in Escherichia coli.
• To elucidate the mechanism by which Cas13a mediates antiphage defense.
• To explore the evolutionary origins of Type VI CRISPR-Cas systems.

Main Methods:

• Expression of LshCas13a in E. coli.
• Analysis of RNA cleavage products using RNA sequencing.
• Assessment of protein synthesis inhibition.
• Investigation of toxin-antitoxin module activation.

Main Results:

• LshCas13a primarily cleaves anticodons in specific transfer RNAs (tRNAs) with uridine-rich anticodons.
• This tRNA cleavage leads to inhibition of protein synthesis, conferring antiphage defense.
• Cas13a-mediated tRNA cleavage indirectly activates mRNA-cleaving RNases from toxin-antitoxin systems, suggesting a backup defense mechanism.

Conclusions:

• Type VI CRISPR-Cas systems, exemplified by LshCas13a, employ tRNA anticodon cleavage as a key mechanism for antiphage defense.
• The observed defense mechanism shares similarities with bacterial anticodon nucleases.
• This suggests that Type VI effectors may have evolved from abortive infection modules containing anticodon nucleases.