Materials and reagents. Purified Cas9 nuclease (S. Pyogenes), BamHI restriction enzyme were purchased from NEB (MA, USA). AmpliScribe T7 High Yield Transcription Kit was purchased from EPICENTRE Biotechnologies (WI, USA). Cell culture related products were purchased from Thermo Fisher Scientific (MA, USA). GoldView, ethidium bromide, agarose gel, DNA and RNA Markers were purchased from Life Technologies (MA, USA). PureLink™ Quick Gel Extraction Kit was purchased from Promega (WI, USA). 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 20×SSC Buffer, formamide, 4% paraformaldehyde, 1% Triton X-100, sodium arsenite, 2.5 mM sodium pyrophosphate and actinomycin D (Act D) were purchased from Sigma Aldrich (MO, USA). 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB), tamoxifen and β-estradiol were purchased from J&K China Chemical (Guangzhou, China). 10×PCR Buffer (without Mg2+), Tris-HCl (100 mM, pH 8.8), 500 mM KCl, 0.8% (v/v) Nonidet, MgCl2 (25mM), dNTP (10mM), 6×DNA Loading Dye, mouse anti-β-actin, rabbit anti-MUC4, IRDye conjugated goat anti-mouse IgG or goat anti-rabbit IgG, Rapid Competent Cell Preps Kit (one step) (SK9307), SanPrep Column Plasmid Mini-Preps Kit (SK8191), Gel Purification Kits (SK8131) and UNIQ-10 Column Trizol Total RNA Purification Kit (SK1321) were purchased from Sangon Biotechnology. (Shanghai, China). MCF-7 (human breast cancer cell line), HeLa (human cervical carcinoma cell line), BRL cells (rat hepatocytes) and HepG2 (human hepatocellular carcinoma cell line) were obtained from the Cell Bank of Central Laboratory at Xiangya Hospital (Changsha, China). MCF-10A cells (normal human mammary epithelial cell line) were purchased from the Cell Bank of the Type Culture Collection of Chinese Academy of Sciences (Beijing, China). Lipofectmine 2000 and Lipofectmine 3000 transfection reagent were purchased from Thermo Fisher Scientific (MA, USA). All other chemicals were of analytical grade and purchased from China National Pharmaceutical Group (Shanghai, China). All solutions and ultrapure water were treated with diethyl pyrocarbonate and autoclaved to prevent RNA degradation.
In vitro transcription and RNA purification. All DNA oligonucleotides (Supplementary Tables 1-3) of HPLC purification were purchased from Sangon Biotechnology (Shanghai, China). gRNAs for in vitro assays were transcribed in vitro by T7 RNA polymerase using linear DNA templates carrying a T7 promoter and complementary sequences for gRNAs. The transcription reaction was prepared by mixing the corresponding linear DNA templates, dithiothreitol, T7 RNA polymerase, RNase inhibitor and NTPs in AmpliScribe T7 reaction buffer. The mixture was transcribed at 37 oC for 2 h and the resulting RNA was purified on a 15% native PAGE gel, excised and stored at -80 oC for future use.
RNA hybridization and gel electrophoresis. In vitro transcribed gRNAs (~200 ng) with or without TK1 RNA oligonucleotides in 10 μL were loaded into a well of a precast agarose gel (1%) stained with GoldView (0.5 μg/mL) and ethidium bromide (EB, 0.5 μg/mL). Electrophoresis was performed at a voltage of 110 V for 60 min. After electrophoresis, the gel was visualized using a Tocan 240 gel imaging system (Tocan Biotechnol., Shanghai, China).
In vitro cleavage assay. Linearized target plasmids were obtained by digesting a plasmid comprising eight tandem repeats for the gRNA target site with PAM (AGG) by the BamHI enzyme. In vitro transcribed gRNAs or agRNAs were annealed by heating to 95 oC, followed by cooling to room temperature. Pre-annealed agRNAs were hybridized with or without TK1 RNA in NEB3 buffer (B7003S, NEB) at room temperature for 1 h. For in vitro cleavage, linearized target plasmids were incubated with Cas9 (100 nM) and gRNAs, agRNAs or hybridized agRNAs in NEB3.1 buffer (B7203S, NEB) at 37 oC for 60 min. To determine the cleavage efficiency of gRNAs with varying extended stem-loop structures, linearized target plasmids were incubated with Cas9 (100 nM) and different gRNAs in NEB3.1 buffer at 37 oC for different times. Reactions were terminated with a 6× DNA loading buffer containing 250 mM EDTA. The mixture was separated by 1% agarose gel electrophoresis and visualized according to the procedure described above.
In vitro gRNA binding and RNase assay. For in vitro binding assay, gRNAs (1 µM) or agRNAs (1 µM) with or without TK1 RNA (1 µM) were incubated with Cas9 protein (1 µM) in NEB3.1 buffer at 37 oC for 30 min. The reactions were terminated with a 6× DNA loading buffer (B548316, Sangon). The mixture was separated by 1% agarose gel electrophoresis and visualized according to the procedure described above.
To test the effect of Cas9 on gRNA protection in an RNase-rich condition in vitro, gRNAs (1 µM) were incubated with Cas9 (1 µM) in NEB3.1 buffer at 37 oC for 30 min to form a binary complex. RNase A (10 mg/ml, Thermo Fisher Scientific, EN0531) was added at 1:500 dilution and the mixture was incubated at 37 oC for 30 min. The reaction was terminated with a 6× DNA loading buffer and the mixture was separated by 1% agarose gel electrophoresis and visualized according to the procedure described above.
Plasmid construction. Plasmids were constructed with standard molecular cloning or Gibson assembly. Plasmids for expressing agRNAs were obtained by cloning the cDNAs for different gRNAs, including sgRNA, gRNA and fgRNA embedded with 3×SRB2, or agRNAs embedded with 3×SRB2, 3×Broccoli, 3×Mango or 3×Pepper into the Addgene plasmid (#60955, a gift from Jonathan Weissman) at the BstxI and XhoI restriction sites under a U6 promoter. Plasmid for expressing a cytosol-localized dCas9 without a nuclear localization signal (NLS) was constructed with a lentiviral vector, and the dCas9 gene was amplified from the Addgene plasmid (#61422, a gift from Feng Zhang).
Plasmid for expressing an exogenous RNA target with a 24×MS2 tag under a miniCMV promoter and MCP-GFP fusion protein under a CMV promoter was constructed with a mammalian expression lentiviral vector. cDNAs of miniCMV promoter, exogenous RNA, 24×MS2 and MCP-GFP were obtained by overlap PCR. cDNAs of miniCMV promoter, exogenous RNA and 24×MS2 were cloned into the KpnI and SpeI sites and the cDNAs of MCP-GFP were cloned into the NheI and ApaI sites.
Plasmids for expressing TK1-responsive agRNAs embedded with n×SRB2 (n = 3, 6, 12, 24) and BFP transfection indicator under a CMV promoter were constructed with a mammalian expression vector pcDNA3.1(+). An mRNA-stabilizing triplex was appended downstream BFP and two HHRs were inserted to release agRNA embedded with n×SRB2 (n = 3, 6, 12, 24). cDNAs of BFP, triplex HHR and agRNA inserted with n×SRB2 (n = 3, 6, 12, 24) were obtained by overlap PCR, purified and cloned into the NheI and ApaI sites in the pcDNA3.1(+). To image exogenous RNA, MUC4 mRNA, β-actin mRNA and lncRNA NEAT1, the cDNAs of the corresponding responsive sequences (Table S3) were obtained and cloned into the plasmid agRNA-24×SRB2 by seamless cloning.
Plasmids for expressing 5S-, 7SK- or CytERM-tagged exogenous RNA with a GFP indicator were constructed with a mammalian expression vector pcDNA3.1(+). The PCR products of GFP were cloned into the NheI and HindIII sites of CMV promotor in the pcDNA3.1(+) vector to generate pcDNA3.1(+)-GFP plasmid. cDNAs of 5S-, 7SK- or CytERM-tagged RNA were obtained by overlap PCR, purified and inserted into the pcDNA3.1(+)-GFP plasmid using seamless cloning. For constructing plasmids expresing G3BP1-GFP and NONO-GFP, cDNAs of G3BP1 and NONO were obtained by overlap PCR and inserted into the pcDNA3.1(+)-GFP plasmid using seamless cloning.
To develop the RNA inducible LUSTER system for transcription activation, the plasmid dCas9-VPR expressing Streptococcus pyogenes dCas9 fused with NLS, VPR and GFP under an EF1α promoter was obtained according to a previous approach using the Addgene plasmid (#61422, a gift from Feng Zhang) as a backbone41. For inducible transcription, DNA sequences with eight tandem repeats for the gRNA target site were obtained with PAM (AGG), miniCMV and the iRFP fluorescent protein by overlap PCR. Plasmid for expressing agRNA and iRFP was obtained by cloning the PCR-generated DNA fragments into the AsiSI and AarI sites in a TK1-responsive agRNA, where the agRNA contains a spacer sequence complementary to the target site.
Cell culture and transfection. HepG2, HeLa, BRL and MCF-7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 50 U/mL penicillin and 50 µg/mL streptomycin in a humidified incubator aerated with 5% CO2 at 37 oC. MCF-10A cells were grown in Mammary Epithelial Cell Growth Medium (MEGM) supplemented with BPE, rhEGF, insulin, hydrocortisone, GA-1000, and 100 ng mL-1 cholera toxin in a humidified incubator aerated with 5% CO2 at 37 oC. For fluorescence imaging, cells were seeded onto sterilized glass coverslips in 35 mm plates with 14-mm wells and grown to ~70% confluency. Cells were transfected with different plasmids as indicated using lipofectamine 3000 transfection agent in an Opti-MEM medium for 4 h before switching to complete medium. Fluorescence images were obtained at a specific time point.
To investigate the stabilization of gRNAs by dCas9 in mammalian cells, MCF-7 cells were transfected with plasmids expressing sgRNA, gRNA and fgRNA with or without cotransfection of a plasmid expressing dCas9 for 24 h. To determine the half-life of gRNAs in live cells, transfected cells were treated with 5 μg/mL Act D to inhibit RNA transcription. Fluorescence images were obtained at different time points.
To image TK1 in different cells, HepG2, MCF-7, HeLa, BRL and MCF-10A cells were transfected with the TK1-responsive LUSTER system expressing agRNA-3×SRB2 and dCas9. To image TK1 at different levels, MCF-7 cells were treated with different doses of β-estradiol or tamoxifen upon transfection with the TK1-responsive LUSTER system.
To investigate the effect of LUSTER system on the subcellular localization of RNAs, MCF-7 cells were cotransfected with plasmid expressing 5S-, 7SK-, or CytERM-tagged RNA together with plasmid expressing agRNA-3×SRB2 responsive to the target RNA.
To study the signal amplification ability of the LUSTER system, MCF-7 cells were cotransfected with plasmids expressing dCas9 and TK1-responsive agRNA embedded with n×SRB2 aptamers (n = 3, 6, 12, 24).
To test the ability of the LUSTER system to image single exogenous RNA in direct comparison with the MS2-MCP approach, MCF-7 cells were cotransfected with plasmids expressing dCas9, exogenous RNA-24×MS2 and agRNA-24×SRB2 responsive to exogenous RNA. To test the ability of our LUSTER system to image single endogenous RNA, MCF-7 cells were transfected with plasmids expressing dCas9 and agRNA-24×SRB2 responsive to a non-repetitive region of MUC4 mRNA.
To image β-actin transcripts and their translocation to SGs, MCF-7 cells were cotransfected with plasmids expressing dCas9, agRNA-24×SRB2 responsive to β-actin and G3BP1-GFP for 24 h. For stress granule induction, transfected cells were incubated with 0.5 mM sodium arsenite at 37 oC. To image lncRNA NEAT1 and its dynamics in paraspeckle organization, MCF-7 cells were cotransfected with plasmids expressing dCas9, agRNA-24×SRB2 responsive to NEAT1 and NONO-GFP for 24 h. Transfected cells were stimulated with arsenite (0.5 mM, 4 h) or DRB (50 µM, 4 h) to regulate NEAT1 expression. Fluorescence images were obtained at different time intervals. For depletion of β-actin or NEAT1, cells were transfected with 0.1 ng of the corresponding siRNAs (Supplementary Table 3) using lipofectamine 2000 according to the manufacturer’s instructions.
To test the ability of RNA inducible transcription, MCF-7 or MCF-10A cells were cotransfected with plasmids expressing dCas9-VPR and agRNA-iRFP-iRFP responsive to TK1 mRNA, fluorescence images were obtained at different times.
Live cell imaging. Transfected cells were washed three times with PBS, and incubated in fresh medium containing tetramethylrhodamine-dinitroaniline (TMR-DN, 1.0 µM), (Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-2-methyl-1-(2,2,2-trifluoroethyl)-1H-imidazol-5(4H)-one (DFHBI-1T, 10 µM), thiazole orange-biotin (TO1-biotin, 10 µM) or HBC620 (1 µM) and 100 mM HEPES, 10 mM MgSO4 at 37 oC for 30 min. Cells were washed and incubated in fresh medium for fluorescence imaging. Fluorescence images were obtained using a 60× oil objective lens (Olympus, Melville, NY) on a confocal laser scanning fluorescence microscope equipped with an Olympus FV1000 confocal scanning system (Nikon, Eclipse TE2000-E). Fluorescence images were acquired using the following collection ranges upon excitation with lasers of different wavelengths. Blue channel: excitation: 405 nm, collection range: 425 - 475 nm, green channel: excitation: 488 nm, collection range: 500 - 550 nm, red channel: excitation: 561 nm, collection range: 575 - 625 nm, NIR channel: excitation: 635 nm, collection range: 650 - 700nm. Fluorescence intensity was analyzed and quantified using the Fiji software. Z-sliced images were obtained through z-axis with 1 μm intervals. For live cell imaging, cells were kept in a humidified chamber aerated with 5% CO2 at 37 oC.
Single molecule RNA FISH. The specificity of the LUSTER system in RNA imaging was verified using the smFISH method according to the literature with slight modifications42. Specifically, primary probes comprising a region for hybridization with Cy5-labeled secondary probes were designed for different target RNAs including 5S-, 7SK- or CytERM- tagged RNA, 24×MS2 tagged exogenous RNA, MUC4, β-actin and NEAT1 (Supplementary Table 4). All probes of HPLC purification were obtained from Sagon Biotech (Shanghai, China). Cells grown in plates with 14-mm glass wells were fixed in a PKM buffer (10 mM Na3PO4, 140 mM KCl and 1 mM MgCl2, pH 7.2) containing 4% paraformaldehyde for 10 min at room temperature and permeabilized in 0.2% Triton X-100 for 10 min. Primary probes (40 pM) were pre-hybridized with Cy5-labeled secondary probes (50 pM) in 1×NEB3 buffer. Pre-hybridized probes in a buffer (10% (w/v) dextran, 10% formamide, 2×SSC in DEPC treated water) were added to the cells and incubated at 37 oC for 16 h. Cells were washed twice with 1×SSC buffer containing 10% formamide at 37 oC for 30 min, twice with PBS for 5 min, followed by staining with Hoechst 33342 (10 μg/ml) and TMR-DN (40-80 nM) in PBS for 5 min. Images were obtained with a Nikon TI-E+A1 SI confocal laser scanning microscope using a 60× oil immersion objective lens.
Quantitative RT-PCR (qRT-PCR) determination of RNA. Concentrations of RNAs including TK1, gRNAs, HHR-cleaved agRNA-3×SRB2, β-actin and NEAT1 under different conditions were determined by qRT-PCR based on a standard curve using GAPDH as an internal reference according to previous studies43. Briefly, cells under different conditions were harvested and counted. Total RNAs were extracted with a UNIQ-10 column Trizol Total RNA Purification Kit according to the manufacturer’s instructions. The purified RNAs were reverse-transcribed into cDNAs with an oligo-dT primer using a RevertAid Premium Reverse Transcriptase Synthesis Kit according to the manufacturer’s instructions. The resulting cDNAs were subjected for qRT-PCR assays using SybrGreen PCR Master Mix on an ABI StepOnePlus qPCR system (CA, USA). Primers for target RNAs and GAPDH were designed with the Primer 5 software (Supplementary Table 5). Ct values for target RNAs and GAPDH were determined and ΔCt values were obtained by subtracting the Ct values for GAPDH from those of target RNAs. Relative levels of target RNAs were calculated using the 2-ΔΔCt method using GAPDH as a reference.
Immunoblotting. Cells were collected, washed twice with cold PBS and lysed with 50 μl of lysis buffer (C500035, Sangon). Total protein concentration was determined with the bicinchoninic acid protein assay. Equal amounts of proteins (30 μg) were separated on 15% SDS polyacrylamide gel and transferred to nitrocellulose membranes. Membranes were blocked with 5% nonfat dry milk and incubated with primary antibodies (mouse anti-β-actin or rabbit anti-MUC4) overnight at 4 oC (1:1000 dilution). After washing three times with PBST (PBS containing 0.1% Tween-20), the membrane was incubated with IRDye conjugated secondary antibodies (goat anti-mouse IgG or goat anti-rabbit IgG, 1:1000 dilution) for 1 h at room temperature. After washing three times with PBST, immunoblots were recorded with Odyssey Infrared Systems (Lincoln, Nebraska, USA). To confirm equal protein loading, tubulin was analyzed.
Flow cytometric analysis. Cells were washed three times with PBS, detached with trypsin, washed twice with PBS and suspended in PBS. Flow cytometry was performed using a FACSVerseTM flow cytometer (BD Biosciences, USA) equipped with 488 nm and 561 nm lasers. At least 10,000 cells were analyzed for each sample. A 488 nm laser was used for GFP excitation and fluorescence emission was collected with a 530/30 nm band-pass filter. A 561 nm laser was used for SRB2 excitation and fluorescence emission was collected with a 610/20 nm band-pass filter. Flow cytometry data were analysed using FlowJo 7.6. For fluorescence quantification, the mean fluorescence intensity of the double-positive population in the red channel was normalized to the mean fluorescence intensity of the same population in the green channel to normalize the red fluorescence to transfection efficiency.
Data and statistical analysis. Fiji was used to analyze microscopic images. Microsoft Excel, OriginPro and GraphPad Prism were used to analyze data and prepare figures for publication. Information on statistical analysis and reproducibility is shown in the corresponding figure legends.
Reporting Summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article.
All data supporting the findings of this study are available within the paper and Supplementary Information. Source data for Figs. 1d, 2d,g-i, 3d-g and 4c,d,g-i, and Extended Data Figs. 2b-d, 5b,d, 6b,d, 7b,c,e-g, 8b, 9b,d, 10a,b,e and Supplementary Figs. 3a-c, 5c,d, 7b-d are provided as source data files. The other datasets generated and analyzed during the current study are available from the corresponding authors upon request.
41. Ying, Z.M., Wang, F., Chu, X., Yu, R.Q. & Jiang, J.H. Activatable CRISPR transcriptional circuits generate functional RNA for mRNA sensing and silencing. Angew. Chem. Int. Ed. Engl.59, 18599‒18604 (2020).
42. Tsanov, N. et al. smiFISH and FISH-quant – a flexible single RNA detection approach with super-resolution capability. Nucleic Acids Res.44, e165 (2016).
43. Nolan, T., Hands, R.E. & Bustin, S.A. Quantification of mRNA using real-time RT-PCR. Nat. Protoc.1, 1559‒1582 (2006).