Programmable RNA targeting with the single-protein CRISPR effector Cas7-11

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• CRISPR-Cas interference relies on Cas nucleases, found in multisubunit (Class 1) or single-protein (Class 2) systems.
• Understanding the diversity and evolution of CRISPR-Cas effectors is crucial for developing new biotechnological tools.

Purpose of the Study:

• To characterize the Cas7-11 effector from the Class 1 CRISPR-Cas system.
• To evaluate its potential as a programmable RNA-targeting tool for gene editing and knockdown.
• To assess its safety and efficacy compared to existing RNA-targeting technologies.

Main Methods:

• Expression and purification of Desulfonema ishimotonii Cas7-11 (DiCas7-11) in E. coli.
• In vitro characterization of DiCas7-11 RNA interference activity, including pre-crRNA processing and target RNA cleavage.
• Engineering DiCas7-11 for RNA knockdown and editing applications in mammalian cells.
• Assessment of cell viability and toxicity of DiCas7-11 compared to shRNA and Cas13.

Main Results:

• DiCas7-11 functions as a single-protein effector within a Class 1 CRISPR-Cas system, derived from a fusion of Cas11 and Cas7 domains.
• DiCas7-11 exhibits potent RNA interference against mRNAs and bacteriophages, processing pre-crRNA and cleaving target RNA specifically.
• Engineered DiCas7-11 demonstrated effective RNA knockdown and editing in mammalian cells with no observed impact on cell viability.
• Cas7-11 showed significantly lower cell toxicity compared to short hairpin RNAs and Cas13.

Conclusions:

• Cas7-11 represents an evolutionary intermediate, transitioning from multisubunit Class 1 effectors to single-protein Class 2 effectors.
• This study expands the known diversity of CRISPR-Cas systems and effector mechanisms.
• Cas7-11 is a promising, non-toxic, and programmable RNA-targeting tool for diverse biotechnological applications, including gene editing and RNA interference.