Cancer Research Institute, School of Basic Medical Sciences, State Key Laboratory of Organ Failure Research, National Clinical Research Center of Kidney Disease, Key Laboratory of Organ Failure Research (Ministry of Education), Southern Medical University, Guangzhou, China.
Beijing National Laboratory for Condensed Matter Physics and Laboratory of Soft Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing, China.
Biotechnol J. 2022 Dec;17(12):e2100381. doi: 10.1002/biot.202100381. Epub 2022 Sep 4.
Endonuclease-deactivated clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease (dCas9) has been repurposed for live-cell imaging of genomic loci. Engineered or evolved dCas9 variants have been developed to increase the applicability of the CRISPR/dCas9 system. However, there have been no systematic comparisons of these dCas9 variants in terms of their performance in the visualization of genomic loci.
Here we demonstrate that dSpCas9 and its variants deSpCas9(1.1), dSpCas9-HF1, devoCas9, and dxCas9(3.7) can be used for CRISPR-based live-cell genomic imaging. dSpCas9 had the greatest utility, with a high labeling efficiency of repetitive sequences-including those with a low number of repeats-and good compatibility with target RNA sequences at the MUC4 locus that varied in length from 13 to 23 nucleotides. We combined CRISPR-Tag with the dSpCas9 imaging system to observe the dynamics of the Tet promoter and found that its movement was restricted when it was active.
These novel Cas9 variants provide a new set of tools for investigating the spatiotemporal regulation of gene expression through live imaging of genomic sites.
内切酶失活的成簇规律间隔短回文重复序列（CRISPR）相关核酸酶（dCas9）已被重新用于基因组位点的活细胞成像。已经开发出工程化或进化的 dCas9 变体，以提高 CRISPR/dCas9 系统的适用性。然而，这些 dCas9 变体在基因组位点可视化方面的性能尚未进行系统比较。
在这里，我们证明 dSpCas9 及其变体 deSpCas9(1.1)、dSpCas9-HF1、devoCas9 和 dxCas9(3.7)可用于基于 CRISPR 的活细胞基因组成像。dSpCas9 的用途最广泛，对重复序列（包括重复次数较少的重复序列）的标记效率很高，并且与 MUC4 基因座的靶 RNA 序列兼容性良好，该序列的长度从 13 到 23 个核苷酸不等。我们将 CRISPR-Tag 与 dSpCas9 成像系统相结合，观察 Tet 启动子的动力学，发现当其活跃时，其运动受到限制。
这些新型 Cas9 变体为通过基因组位点的活细胞成像研究基因表达的时空调控提供了一组新工具。
