CRISPR/Pepper‐tDeg: A Live Imaging System Enables Non‐Repetitive Genomic Locus Analysis with One Single‐Guide RNA

Author:

Chen Meng1,Huang Xing1,Shi Yakun1,Wang Wen2,Huang Zhan3,Tong Yanli1,Zou Xiaoyong3,Xu Yuzhi4ORCID,Dai Zong1ORCID

Affiliation:

1. Guangdong Provincial Key Laboratory of Sensing Technology and Biomedical Instrument School of Biomedical Engineering Shenzhen Campus of Sun Yat‐Sen University Sun Yat‐Sen University Shenzhen 518107 China

2. School of Pharmaceutical Sciences Sun Yat‐Sen University Guangzhou 510275 China

3. School of Chemistry Sun Yat‐Sen University Guangzhou 510275 China

4. Scientific Research Center The Seventh Affiliated Hospital Sun Yat‐Sen University Shenzhen 518107 China

Abstract

AbstractCRISPR‐based genomic‐imaging systems have been utilized for spatiotemporal imaging of the repetitive genomic loci in living cells, but they are still challenged by limited signal‐to‐noise ratio (SNR) at a non‐repetitive genomic locus. Here, an efficient genomic‐imaging system is proposed, termed CRISPR/Pepper‐tDeg, by engineering the CRISPR sgRNA scaffolds with the degron‐binding Pepper aptamers for binding fluorogenic proteins fused with Tat peptide derived degron domain (tDeg). The target‐dependent stability switches of both sgRNA and fluorogenic protein allow this system to image repetitive telomeres sensitively with a 5‐fold higher SNR than conventional CRISPR/MS2‐MCP system using “always‐on” fluorescent protein tag. Subsequently, CRISPR/Pepper‐tDeg is applied to simultaneously label and track two different genomic loci, telomeres and centromeres, in living cells by combining two systems. Given a further improved SNR by the split fluorescent protein design, CRISPR/Pepper‐tDeg system is extended to non‐repetitive sequence imaging using only one sgRNA with two aptamer insertions. Neither complex sgRNA design nor difficult plasmid construction is required, greatly reducing the technical barriers to define spatiotemporal organization and dynamics of both repetitive and non‐repetitive genomic loci in living cells, and thus demonstrating the large application potential of this genomic‐imaging system in biological research, clinical diagnosis and therapy.

Funder

National Natural Science Foundation of China

Shenzhen Fundamental Research Program

Publisher

Wiley

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