dCas13-mediated translational repression for accurate gene silencing in mammalian cells

Antonios Apostolopoulos, Naohiro Kawamoto, Siu Yu A. Chow, Hitomi Tsuiji, Yoshiho Ikeuchi, Yuichi Shichino () and Shintaro Iwasaki ()
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Antonios Apostolopoulos: The University of Tokyo
Naohiro Kawamoto: RIKEN Cluster for Pioneering Research
Siu Yu A. Chow: The University of Tokyo
Hitomi Tsuiji: Aichi Gakuin University
Yoshiho Ikeuchi: The University of Tokyo
Yuichi Shichino: RIKEN Cluster for Pioneering Research
Shintaro Iwasaki: The University of Tokyo

Nature Communications, 2024, vol. 15, issue 1, 1-18

Abstract: Abstract Current gene silencing tools based on RNA interference (RNAi) or, more recently, clustered regularly interspaced short palindromic repeats (CRISPR)‒Cas13 systems have critical drawbacks, such as off-target effects (RNAi) or collateral mRNA cleavage (CRISPR‒Cas13). Thus, a more specific method of gene knockdown is needed. Here, we develop CRISPRδ, an approach for translational silencing, harnessing catalytically inactive Cas13 proteins (dCas13). Owing to its tight association with mRNA, dCas13 serves as a physical roadblock for scanning ribosomes during translation initiation and does not affect mRNA stability. Guide RNAs covering the start codon lead to the highest efficacy regardless of the translation initiation mechanism: cap-dependent, internal ribosome entry site (IRES)-dependent, or repeat-associated non-AUG (RAN) translation. Strikingly, genome-wide ribosome profiling reveals the ultrahigh gene silencing specificity of CRISPRδ. Moreover, the fusion of a translational repressor to dCas13 further improves the performance. Our method provides a framework for translational repression-based gene silencing in eukaryotes.

Date: 2024
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DOI: 10.1038/s41467-024-46412-7

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