Department of Biochemistry and Biophysics, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642, USA; Center for RNA Biology, University of Rochester, Rochester, NY 14642, USA.
Center for RNA Biology, University of Rochester, Rochester, NY 14642, USA; Department of Biomedical Genetics, School of Medicine and Dentistry, University of Rochester, NY 14642, USA.
Methods. 2022 Oct;206:58-68. doi: 10.1016/j.ymeth.2022.08.007. Epub 2022 Aug 17.
Prokaryotic adaptive immune systems use Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and CRISPR Associated (Cas) proteins to target and cleave foreign genetic elements in an RNA-guided manner [1-3]. Type VI CRISPR-Cas systems contain a single effector ribonuclease, Cas13, that binds and processes a CRISPR-RNA (crRNA; also known as a guide-RNA), forming an RNA-guided RNA-targeting effector complex [4,5]. Previous studies have shown that Cas13 can be engineered to target and modulate RNA processes in human cells, illustrating the versatility and specificity of Cas13 as an RNA knockdown (KD), splicing, editing, or imaging tool [6-8]. While Cas13 has been successfully used by several groups, our lab has observed significant variability in Cas13 KD ability depending which protocol is being followed [9-12]. To further understand this variability and generate a robust Cas13 KD protocol we thoroughly tested which Cas13 ortholog to use, the duration of KD experiments, the amount of plasmid DNA transfected, methods for analyzing KD efficiency, and report an optimized method for carrying out and analyzing Cas13 mediated RNA KD experiments. The method outlined in this paper illustrates a faster and more reliable protocol to iteratively test gRNA performance and target gene KD.
原核适应性免疫系统利用规律成簇间隔短回文重复序列 (CRISPRs) 和 CRISPR 相关 (Cas) 蛋白以 RNA 指导的方式靶向和切割外来遗传元件[1-3]。VI 型 CRISPR-Cas 系统包含单个效应核糖核酸酶 Cas13，它结合并处理 CRISPR-RNA（也称为指导 RNA），形成 RNA 引导的 RNA 靶向效应复合物[4,5]。先前的研究表明，Cas13 可以被工程化为靶向和调节人类细胞中的 RNA 过程，这说明了 Cas13 作为 RNA 敲低 (KD)、剪接、编辑或成像工具的多功能性和特异性[6-8]。虽然 Cas13 已被多个研究小组成功使用，但我们实验室观察到，根据所遵循的方案不同，Cas13 KD 能力存在显着差异[9-12]。为了进一步了解这种可变性并生成稳健的 Cas13 KD 方案，我们彻底测试了要使用的 Cas13 同源物、KD 实验的持续时间、转染的质粒 DNA 量、分析 KD 效率的方法，并报告了一种优化的 Cas13 介导的 RNA KD 实验的执行和分析方法。本文中概述的方法说明了一种更快、更可靠的方案，可用于迭代测试 gRNA 性能和靶基因 KD。
