Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, OX3 9DS, UK.
Weatherall Institute of Molecular Medicine, MRC Molecular Haematology Unit, NIHR Oxford Biomedical Research Centre Programme, University of Oxford, Oxford, OX3 9DS, UK.
Nat Commun. 2019 Apr 2;10(1):1490. doi: 10.1038/s41467-019-09148-3.
Spatial/temporal control of Cas9 guide RNA expression could considerably expand the utility of CRISPR-based technologies. Current approaches based on tRNA processing offer a promising strategy but suffer from high background. Here, to address this limitation, we present a screening platform which allows simultaneous measurements of the promoter strength, 5', and 3' processing efficiencies across a library of tRNA variants. This analysis reveals that the sequence determinants underlying these activities, while overlapping, are dissociable. Rational design based on the ensuing principles allowed us to engineer an improved tRNA scaffold that enables highly specific guide RNA production from a Pol-II promoter. When benchmarked against other reported systems this tRNA scaffold is superior to most alternatives, and is equivalent in function to an optimized version of the Csy4-based guide RNA release system. The results and methods described in this manuscript enable avenues of research both in genome engineering and basic tRNA biology.
Cas9 引导 RNA 表达的时空控制可以极大地扩展基于 CRISPR 的技术的应用。目前基于 tRNA 加工的方法提供了一种很有前途的策略，但存在背景高的问题。在这里，为了解决这个限制，我们提出了一个筛选平台，允许在 tRNA 变体文库中同时测量启动子强度、5' 和 3' 加工效率。该分析表明，虽然这些活性的序列决定因素重叠，但可以分离。基于后续原理的合理设计使我们能够设计出一种改良的 tRNA 支架，从而能够从 Pol-II 启动子高效特异性地产生引导 RNA。与其他报道的系统相比，该 tRNA 支架优于大多数替代方案，并且在功能上与基于 Csy4 的引导 RNA 释放系统的优化版本相当。本文描述的结果和方法为基因组工程和基本 tRNA 生物学的研究开辟了途径。
