Programmable modulation of ribosomal frameshifting by mRNA targeting CRISPR-Cas12a system

Area of Science:

• Molecular Biology
• Genetics
• Virology

Background:

• Minus 1 programmed ribosomal frameshifting (-1 PRF) is a crucial translational regulation conserved across species.
• This process is essential for the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
• Targeting the RNA structures involved in -1 PRF offers therapeutic potential.

Purpose of the Study:

• To investigate the role of CRISPR RNA and CRISPR-Cas12a in modulating -1 PRF.
• To explore the potential of CRISPR-Cas12a as a tool to disrupt SARS-CoV-2 replication.
• To develop novel strategies for antiviral therapies against coronaviruses.

Main Methods:

• Utilizing CRISPR RNA to stimulate -1 PRF.
• Employing CRISPR-Cas12a and CRISPR-Cas9 complexes to assess their impact on -1 PRF efficiency.
• Performing CRISPR-Cas12a targeting screens to identify effective sites for disrupting SARS-CoV-2 frameshifting pseudoknots.
• Quantifying -1 PRF suppression in vitro and in mammalian cells.

Main Results:

• CRISPR RNA was shown to stimulate -1 PRF.
• CRISPR-Cas12a, unlike CRISPR-Cas9, enhanced -1 PRF by stalling ribosomes.
• Targeting SARS-CoV-2 pseudoknot structures with CRISPR-Cas12a resulted in significant suppression of -1 PRF (>70% in vitro, ~50% in cells).

Conclusions:

• The CRISPR-Cas12 system can modulate -1 PRF efficiency by stalling ribosomes and deforming RNA structures.
• CRISPR-Cas12a represents a promising new strategy for developing antiviral therapies against coronaviruses.
• This research expands the functional applications of CRISPR systems in translational regulation and pandemic preparedness.