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

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 (CRISPRCas13). Thus, a more specific method of gene knockdown is needed. Here, we developed "CRISPR{delta}", 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 CRISPR{delta}. 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.