CRISPR-Cas9을 이용한 살아있는 세포에서의 mRNA와 단백질의 동시 시각화 기법

Abstract

mRNA and proteins play critical roles as spatiotemporal steps maintaining cell homeostasis and regulating biosynthesis. Proteins are important as catalytic materials for various chemical reactions in cells. Thus, detecting mRNA and protein is necessary to understand their regulation. However, there is currently no technology to simultaneously image mRNA and protein in living cells. In this study, a heat shock transcription factor (HSF) 1 donor DNA knock-in cell line was developed using CRISPR-Cas9 to image HSF1 mRNA and HSF1 protein at the same time. A HSF1 knock-in cell line was prepared by inserting donor DNA by homologous recombination around the HSF1 stop codon. Specifically, HSF1 donor DNA contained homologous arms of appropriate length on both sides. EGFP was used for protein tagging, and a puromycin resistance cassette was the selection marker. I also applied the MS2 RNA-labeling system in donor DNA. Live imaging of mRNA dynamics at a single RNA molecule resolution was achieved in live cells using this binary system to express mRNA of the target tagged with 12 copies of RNA hairpin MS2-binding site and fluorescent MS2 bacteriophage coated protein (MCP). In addition, I designed single-guide RNA with low off-target and high cleavage efficiency. A stable HSF1 cell line was constructed using CRISPR-Cas9, and the HSF1 protein was visualized through an enhanced green fluorescent protein signal. Furthermore HSF1 mRNA was visualized in the cytoplasm by exogenously expressing fluorescent MCPs. This system provides a new technique for visualizing specific mRNA and protein to study dynamic changes in endogenous mRNAs and proteins in living cells.