Biomanufacturing and Biomaterials Department, Sandia National Laboratories, Livermore, CA, USA.
Deconstruction Division, Joint BioEnergy Institute, Lawrence Berkeley National Laboratory, Emeryville, CA, USA.
Nucleic Acids Res. 2022 Aug 26;50(15):8986-8998. doi: 10.1093/nar/gkac680.
Tools for synthetically controlling gene expression are a cornerstone of genetic engineering. CRISPRi and CRISPRa technologies have been applied extensively for programmable modulation of gene transcription, but there are few such tools for targeted modulation of protein translation rates. Here, we employ CRISPR-Cas13 as a programmable activator of translation. We develop a novel variant of the catalytically-deactivated Cas13d enzyme dCasRx by fusing it to translation initiation factor IF3. We demonstrate dCasRx-IF3's ability to enhance expression 21.3-fold above dCasRx when both are targeted to the start of the 5' untranslated region of mRNA encoding red fluorescent protein in Escherichia coli. Activation of translation is location-dependent, and we show dCasRx-IF3 represses translation when targeted to the ribosomal binding site, rather than enhancing it. We provide evidence that dCasRx-IF3 targeting enhances mRNA stability relative to dCasRx, providing mechanistic insights into how this new tool functions to enhance gene expression. We also demonstrate targeted upregulation of native LacZ 2.6-fold, showing dCasRx-IF3's ability to enhance expression of endogenous genes. dCasRx-IF3 requires no additional host modification to influence gene expression. This work outlines a novel approach, CRISPR-RNAa, for post-transcriptional control of translation to activate gene expression.
基因表达的合成调控工具是基因工程的基石。CRISPRi 和 CRISPRa 技术已被广泛应用于可编程调节基因转录，但很少有这样的工具用于靶向调节蛋白质翻译率。在这里，我们将 Cas13 用作翻译的可编程激活剂。我们通过将其融合到翻译起始因子 IF3 上来开发一种新型的失活 Cas13d 酶 dCasRx 变体。我们证明了当 dCasRx-IF3 靶向大肠杆菌中编码红色荧光蛋白的 mRNA5'非翻译区的起始子时，其表达水平比 dCasRx 高 21.3 倍。翻译的激活是位置依赖性的，我们表明当靶向核糖体结合位点时，dCasRx-IF3 会抑制翻译，而不是增强翻译。我们提供的证据表明，与 dCasRx 相比，dCasRx-IF3 靶向增强了 mRNA 的稳定性，这为该新工具如何增强基因表达的机制提供了见解。我们还证明了靶向上调内源性 LacZ 2.6 倍，表明 dCasRx-IF3 能够增强内源性基因的表达。dCasRx-IF3 无需额外的宿主修饰即可影响基因表达。这项工作概述了一种新的方法，即 CRISPR-RNAa，用于翻译的转录后控制以激活基因表达。
