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

Shih-Hong Huang; Shih-Cheng Chen; Tsu-Ying Wu; Cheng-Yao Chen; Chien-Hung Yu

doi:10.1016/j.isci.2023.108492

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

iScience. 2023 Nov 19;26(12):108492. doi: 10.1016/j.isci.2023.108492. eCollection 2023 Dec 15.

Authors

Shih-Hong Huang^{1

2}, Shih-Cheng Chen^{1

3}, Tsu-Ying Wu⁴, Cheng-Yao Chen^{4

5}, Chien-Hung Yu^{1

2}

Affiliations

¹ Department of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
² Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
³ National Institute of Cancer Research, National Health Research Institutes, Tainan, Taiwan.
⁴ YD BioLabs, Inc., Hsinchu, Taiwan.
⁵ School of Medical Laboratory Science and Biotechnology, Taipei Medical University, Taipei, Taiwan.

Abstract

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: Biotechnology; Molecular biology; Virology.