Super-resolution imaging of fluorescently labeled, endogenous RNA Polymerase II in living cells with CRISPR/Cas9-mediated gene editing.

Won-Ki Cho,Yoon J Jung,Susan Mullen,Ibrahim I Cissé,Tzer Han Tan,Namrata Jayanth

doi:10.1038/srep35949

Abstract

Live cell imaging of mammalian RNA polymerase II (Pol II) has previously relied on random insertions of exogenous, mutant Pol II coupled with the degradation of endogenous Pol II using a toxin, α-amanitin. Therefore, it has been unclear whether over-expression of labeled Pol II under an exogenous promoter may have played a role in reported Pol II dynamics in vivo. Here we label the endogenous Pol II in mouse embryonic fibroblast (MEF) cells using the CRISPR/Cas9 gene editing system. Using single-molecule based super-resolution imaging in the living cells, we captured endogenous Pol II clusters. Consistent with previous studies, we observed that Pol II clusters were short-lived (cluster lifetime ~8 s) in living cells. Moreover, dynamic responses to serum-stimulation, and drug-mediated transcription inhibition were all in agreement with previous observations in the exogenous Pol II MEF cell line. Our findings suggest that previous exogenously tagged Pol II faithfully recapitulated the endogenous polymerase clustering dynamics in living cells, and our approach may in principle be used to directly label transcription factors for live cell imaging.

Highlights

RNA Polymerase II (Pol II) is the molecular complex responsible for the synthesis of all messenger RNAs, as well as some non-coding RNAs in all eukaryotes[1,2,3,4,5]
About the CRISPR/Cas[9] design, to induce Cas9-mediated DNA double-strand break (DSB) near the start codon of the Rpb[1] gene, the first exon and a part of the first intron were targeted with single guide RNAs that confer specificity to the Cas[9] nuclease (Fig. 1a and Supplementary Fig. 1a)
Each single guide RNAs (sgRNAs) was cloned into a Streptococcus pyogenes Cas[9] (SpCas9) vector with a distinct promoter to co-express the sgRNAs and Cas[9] in vivo (Supplementary Fig. 1b)[16]

Summary

Introduction

RNA Polymerase II (Pol II) is the molecular complex responsible for the synthesis of all messenger RNAs, as well as some non-coding RNAs in all eukaryotes[1,2,3,4,5]. Previous approaches in mammalian cells relied on the ability to render exogenous Pol II resistant to a toxin, α-amanitin that degrades non-mutant (endogenous) Pol II. Stable cell lines can be created where endogenous Pol II is degraded and replaced by labeled Pol II, often expressed under a strong exogenous promoter, and randomly inserted in the genome[10,12]. It was unclear whether genome insertion context or the uncontrolled expression of exogenous Pol II may have played a significant role in the protein dynamics observed. We investigated the organization and dynamics of the labeled, endogenous Pol II using single-molecule based super-resolution imaging[10,12]

Methods

Results

Conclusion