dCas13-mediated translational repression for accurate gene silencing in mammalian cells
Antonios Apostolopoulos1,2, Naohiro Kawamoto2, Siu Yu A Chow3
1Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba, 277-8561, Japan.
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.
Insights
Researchers developed CRISPRδ, a novel gene silencing method that blocks translation without degrading mRNA. This highly specific approach offers a precise alternative to RNA interference and CRISPR-Cas13 systems for gene knockdown.
Area of Science:
- Molecular Biology
- Gene Regulation
- Biotechnology
Background:
- Current gene silencing methods like RNA interference (RNAi) and CRISPR-Cas13 have limitations, including off-target effects and collateral mRNA cleavage.
- There is a need for more specific and effective gene knockdown strategies.
Purpose of the Study:
- To develop a novel gene silencing tool with enhanced specificity and reduced off-target effects.
- To establish a method for translational silencing using catalytically inactive Cas13 (dCas13).
Main Methods:
- CRISPRδ system utilizing catalytically inactive Cas13 (dCas13) proteins and guide RNAs targeting the start codon.
- Assessment of translational silencing efficacy across different translation initiation mechanisms (cap-dependent, IRES, RAN).
- Genome-wide ribosome profiling to evaluate gene silencing specificity.
Main Results:
- CRISPRδ effectively silences gene translation by acting as a physical roadblock for ribosomes.
- High silencing efficacy was observed when guide RNAs targeted the start codon, irrespective of the translation initiation mechanism.
- Genome-wide ribosome profiling confirmed the ultrahigh specificity of CRISPRδ, with minimal impact on mRNA stability.
- Fusion of dCas13 with a translational repressor further enhanced silencing performance.
Conclusions:
- CRISPRδ offers a highly specific and effective method for gene knockdown by inhibiting translation.
- This approach overcomes the limitations of existing RNAi and CRISPR-Cas13 systems.
- CRISPRδ provides a versatile framework for translational repression-based gene silencing in eukaryotic systems.
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