Why the study?
Existing gene silencing tools like RNAi and CRISPR-Cas13 have critical drawbacks, including off-target effects and collateral mRNA cleavage, creating a need for a more specific gene knockdown method.
Does CRISPRδ improve gene knockdown specificity and efficacy in mammalian cells?
Does CRISPRδ improve gene knockdown specificity and efficacy in mammalian cells?
CRISPRδ provides a highly specific framework for translational repression-based gene silencing in eukaryotes, overcoming off-target effects of RNAi and collateral cleavage of Cas13.
CRISPRδ may enable more specific gene knockdown; leaves open clinical translation pending validation in disease models.
Summary 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 (CRISPR⃩Cas13). Thus, a more specific method of gene knockdown is needed. Here, we developed “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 or internal ribosome entry site (IRES)-dependent translation. Strikingly, genome-wide ribosome profiling revealed the extremely high gene knockdown specificity of CRISPRδ. Moreover, fusion of a translational repressor to dCas13 ensured further improvement of the knockdown efficacy. Our method provides a framework for translational repression-based gene silencing in eukaryotes.
No takes yet. Share an insight, caveat, or question.
Apostolopoulos et al. (2023) studied this question.
Synapse has enriched 5 closely related papers on similar clinical questions. Consider them for comparative context: