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

Abstract

Summary: Minus 1 programmed ribosomal frameshifting (−1 PRF) is a conserved translational regulation event essential for critical biological processes, including the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication. Efficient trans-modulation of the structured RNA element crucial to −1 PRF will endow the therapeutic application. Here, we demonstrate that CRISPR RNA can stimulate efficient −1 PRF. Assembled CRISPR-Cas12a, but not CRISPR-Cas9, complex further enhances −1 PRF efficiency through its higher capacity to stall translating ribosomes. We additionally perform CRISPR-Cas12a targeting to impair the SARS-CoV-2 frameshifting pseudoknot structure via a focused screening. We demonstrate that targeting CRISPR-Cas12a results in more than 70% suppression of −1 PRF in vitro and about 50% suppression in mammalian cells. Our results show the expanded function of the CRISPR-Cas12 system in modulating −1 PRF efficiency through stalling ribosomes and deforming frameshifting stimulatory signals, which could serve as a new strategy for future coronavirus pandemics.

Keywords

• Molecular biology
• Virology
• Biotechnology