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.

当前基于 RNA 干扰 (RNAi) 或最近的基于聚类规则间隔短回文重复序列 (CRISPR)-Cas13 系统的基因沉默工具具有严重缺陷，例如脱靶效应 (RNAi) 或附带 mRNA 裂解 (CRISPR-Cas13) 。因此，需要一种更具体的基因敲低方法。在这里，我们开发了 CRISPRδ，一种利用催化失活 Cas13 蛋白 (dCas13) 进行翻译沉默的方法。由于 dCas13 与 mRNA 紧密相关，因此它可以作为翻译起始过程中扫描核糖体的物理障碍，并且不会影响 mRNA 的稳定性。无论翻译起始机制如何：帽依赖性、内部核糖体进入位点 (IRES) 依赖性或重复相关的非 AUG (RAN) 翻译，覆盖起始密码子的向导 RNA 都能实现最高功效。引人注目的是，全基因组核糖体分析揭示了 CRISPRδ 的超高基因沉默特异性。此外，翻译抑制子与 dCas13 的融合进一步提高了性能。我们的方法为真核生物中基于翻译抑制的基因沉默提供了框架。