A programmable CRISPR-based light-up RNA stabilizer for fluorogenic RNA imaging

doi:10.21203/rs.3.rs-1435078/v1

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A programmable CRISPR-based light-up RNA stabilizer for fluorogenic RNA imaging

https://doi.org/10.21203/rs.3.rs-1435078/v1

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Live cell imaging of RNAs and dynamics is crucial for understanding their functions and pathological roles. However, fluorogenic imaging of endogenous RNAs remains a challenge. Here, we report the design of a programmable CRISPR-based light-up RNA stabilizer (LUSTER) that enables fluorogenic imaging of endogenous RNAs without the need for genetically inserted tags. We show that dCas9 can bind and stabilize the otherwise degraded extended guide RNAs (gRNAs). A light-up aptamer-embedded, mis-folded gRNA is designed to restore the dCas9-binding conformation in response to target RNAs and to provide fluorogenic signals from the dCas9-stabilized gRNA for RNA imaging. Our system is universally applicable for quantitative detection and dynamic imaging of diverse RNA targets, including mRNA detection, single-molecule RNA imaging, cytosolic or nucleic RNA localization imaging, and RNA-induced transcriptional activation in mammalian cells. The programmability and general applicability of the LUSTER system highlight its broad utility in RNA biology and theranostics.

RNAs play crucial roles in numerous cellular processes with complicated dynamics in expression, modifications, translocation, and degradation. Live cell imaging of RNA abundance, localization and dynamics has revolutionized our ability to elucidate underlying biological mechanisms and provide insights for disease theranostics. Though there are a diverse set of techniques available for detecting RNAs in vitro or in fixed cells, direct detection and imaging of RNAs are more challenging and largely rely on two strategies: multimeric protein-binding RNA tags (MS2 or PP7 motifs) that recruit fluorescent protein fusions^1,2, and fluorescence self-quenched hybridization probes^3,4. The multimeric RNA tag system generally involves laborious genetic engineering of target RNAs and suffers significant background signals from unbound fluorescent proteins. The hybridization probes, on the other hand, are limited by their delivery issues and the degradation-associated background fluorescence.

New approaches have emerged for live cell imaging of RNA localization and dynamics. A prominent advance is the light-up RNA aptamers, in vitro selected RNA sequences that can bind to cognate non-fluorescent dyes and induce fluorescence enhancement^5-12. Genetic incorporation of fluorogenic aptamer tags into target RNAs allows direct visualization of their expression and localization^13-16. Significant improvements using RNA aptamer stabilized fluorescent proteins have provided a completely genetically encodable tool for live cell imaging of target RNAs^17,18. Unfortunately, most fluorogenic aptamer systems still require genetic insertion of aptamer motifs into target RNAs, precluding direct imaging for endogenous RNAs. Additionally, these systems generally exhibited inferior brightness in mammalian cells due to their limited folding efficiency, compromising the detection of low-abundance RNAs. Another important advance is the clustered regularly-interspaced short palindromic repeats (CRISPR) system¹⁹ for targeted RNA imaging. Recent studies have demonstrated a fluorescent protein fused nuclease-deficient Cas9 (dCas9) system for programmable targeting and visualization of high-abundance RNAs with the aid of synthetic PAMmer oligonucleotides²⁰. More recently, labeling and tracking of RNAs has been achieved with the CRISPR/dCas13 system, using guide RNAs (gRNAs) for programmable RNA targeting and dCas13-fluorescent protein fusions for imaging²¹. These CRISPR systems afford the advantage of direct imaging of endogenous RNAs. However, they are not fluorogenic and exhibit a limited signal-to-background ratio due to the unbound dCas9/13-fluorescent protein fusions. Approaches that enable fluorogenic imaging of endogenous RNAs without genetically inserted tags remain a challenge.

Here, we propose a new concept for the programmable CRISPR-based light-up RNA Stabilizer (LUSTER) system that enables direct fluorogenic imaging of endogenous RNAs without the need for genetically inserted tags. We show that dCas9 can strongly bind to a fully extended gRNA and stabilize it against degradation in vitro or in mammalian cells. By optimizing different light-up aptamer-embedded gRNAs with misfolded conformations, we develop a gRNA design that can restore the dCas9-binding conformation in response to target RNAs, affording stabilized light-up aptamers for fluorogenic RNA imaging. This system is successfully demonstrated for mRNA detection, cytosolic mRNA translocation imaging, nucleic lncRNA localization, single-molecule RNA imaging, and RNA-induced transcriptional activation in mammalian cells. We expect that the LUSTER system can provide a broadly useful, programmable platform for RNA studies and RNA-activated genome engineering.

Cas9 stabilizes gRNA with embedded light-up RNA. Most of light-up aptamers exhibit limited stability in mammalian cells, and embedding these aptamers in a stabilizing scaffold is necessary for their high expression and protection from degradation. In CRISPR system, Cas9/dCas9 protein can bind to gRNA, driving significant conformational changes within Cas9/dCas9 and forming a binary Cas9/dCas9−gRNA complex^22,23. However, evidence for Cas9/dCas9−gRNA complex in mammalian cells is still lacking. We envisioned that extension of gRNA could improve the specific Cas9/dCas9−gRNA recognition, allowing protect the gRNA scaffold with embedded light-up aptamers from degradation and affording stabilized fluorogenic Cas9/dCas9−gRNA complex in mammalian cells. Motivated by this hypothesis, we designed three gRNAs with varying extension of stem-loop structures (Fig. 1a and Supplementary Table 1), a short-version gRNA (sgRNA)²⁴, an extended gRNA (egRNA)²⁵ and a fully extended gRNA (fgRNA), and performed gel electrophoresis to identify the optimal gRNA structure stabilized by Cas9/dCas9. All of these gRNAs were protected from ribonuclease A (RNase A) degradation in the presence of Cas9, with fgRNA displaying the strongest protection (Extended Data Fig. 1a,b). The Cas9-mediated protection of gRNAs was ascribed to the formation of stable binary Cas9–gRNA complexes (Extended Data Fig. 1c), with the best association achieved for fgRNA. Strikingly, the Cas9–fgRNA complex exhibited a much higher rate in target DNA cleavage than the sgRNA and egRNA counterparts (Extended Data Fig. 1d), indicating that better association with gRNA was likely to enhance Cas9 activity in DNA targeting and cleavage.

To validate Cas9/dCas9-mediated protection of gRNA in live mammalian cells, we engineered three gRNAs with a tandem light-up aptamer, 3×SRB2⁹, embedded in the tetra-loop (Extended Data Fig. 2a and Supplementary Table 1) and a cytosol-localized dCas9 protein by deleting the nuclear localization signal (NLS) (Supplementary Fig. 1). We found that MCF-7 cells expressing only gRNAs without dCas9 exhibited negligible fluorescence upon incubation of the cognate dye tetramethylrhodamine-dinitroaniline (TMR-DN) (Fig. 1b, Extended Data Fig. 2b and Supplementary Fig. 2), consistent with previous reports that gRNAs were highly unstable in mammalian cells^{26 − 28}. In contrast, cells with dCas9 co-expression delivered bright fluorescence from the light-up aptamers. Flow cytometry and qRT-PCR testified much higher expression of these three gRNAs under dCas9 co-expression, with the highest expression obtained for fgRNA (Fig. 1c and Extended Data Fig. 2c,d). Further investigation of gRNA stability was performed by treating cells with actinomycin D (Act D) to inhibit RNA transcription. Co-expression of dCas9 and gRNAs delivered strong and stabilized fluorescence signals (50% of the initial fluorescence retained over 4.0 h), and fgRNA produced the most stabilized fluorescence with a half-life up to ~ 6 h (Fig. 1d). These results suggested that dCas9–gRNA recognition could protect gRNAs with embedded SRB2 aptamer from degradation in mammalian cells, and the stability increased for gRNAs with stronger association to dCas9. These results also supported a previous finding of target DNA-dependent protection of gRNA^{26 − 28}, in which stronger protection was observed in the ternary Cas9–gRNA–DNA complex. More importantly, the discovery of gRNA protection by Cas9/dCas9 alone inspired us to develop the LUSTER system for fluorogenic RNA imaging.

Design of an RNA-responsive gRNA. We sought to insert two complementary regions into the gRNA such that these regions formed stable intramolecular hybridization. We hypothesized that this intramolecular hybridization could disrupt gRNA’s conformation and abrogate its binding to Cas9/dCas9, and strand displacement mediated by target RNA could restore its Cas9/dCas9-binding conformation. We designed the RNA-responsive gRNA from fgRNA due to its improved association to Cas9/dCas9 using a model target TK1, an mRNA overexpressed in cancer cells²⁹. Previous work demonstrated that the three regions (tetra-loop, stem-loop 2 and 3’ terminus) of gRNA in the binary Cas9/dCas9-gRNA complex were solvent-exposed and tolerant to sequence variations^22,23. Therefore, we tested three designs of abrogated gRNAs (agRNAs) by incorporating intramolecular hybridization between two of the three candidate regions, and optimized the complementary lengths from 11 to 14 bp (Fig. 1e and Supplementary Table 2). In vitro DNA cleavage assay using gel electrophoresis revealed that four agRNAs, with 13-bp and 14-bp complementation between stem-loop 2 and the tetra-loop or 3’ terminus, completely abolished the cleavage activity, whereas the other agRNAs still retained substantial cleavage activity (Fig. 1f). This result suggested a crucial role of stem-loop 2 in gRNA for Cas9 binding, as these agRNAs with fully abolished activity all involved its intramolecular complementation.

We investigated the responsiveness of these four agRNAs to TK1 mRNA, and found that two agRNAs with 13-bp complementary regions between stem-loop 2 and the tetra-loop or 3’ terminus exhibited high cleavage activity upon mRNA hybridization (Fig. 1g). We chose the agRNA with 13-bp complementary regions between stem-loop 2 and the tetra-loop as the optimal agRNA for further study, considering that both complementary regions were internal domains of the gRNA with potentially improved stability in live cells³⁰. A further assay for the selected agRNA showed that its fully abolished activity was attributed to disrupted binding to Cas9, and hybridization with TK1 mRNA restored its binding ability (Extended Data Fig. 3a). Besides, the restored Cas9 activity was dynamically dependent upon TK1 concentration (Extended Data Fig. 3b) and specific to TK1–agRNA hybridization (Extended Data Fig. 3c). Together, these results suggested that the optimized design of agRNA could abrogate its binding to Cas9/dCas9, enabling specific RNA-responsive control of the CRISPR system.

Live cell imaging of TK1 mRNA. We exploited the RNA-responsive gRNA design to develop the LUSTER imaging system using two plasmid constructs: one for the agRNA embedded with a 3×SRB2 array (agRNA–3×SRB2, Supplementary Table 3) under a U6 promoter and the other for the cytosol-localized dCas9 under an EF1α promoter (Fig. 2a,b and Supplementary Fig. 3). In the absence of target RNAs, the agRNA–3×SRB2 was in misfolded conformation and unable to interact with dCas9, leading to rapid degradation and diminished fluorescence for agRNA–3×SRB2. After hybridization with target RNAs, the agRNA–3×SRB2 restored its dCas9-binding conformation, stabilizing the light-up RNA embedded gRNA against degradation with a fluorogenic signal for imaging.

We then explored the LUSTER system for TK1 mRNA imaging in live cells. Co-expression of agRNA–3×SRB2 and dCas9 delivered negligible fluorescence in TK1-negative MCF-10A cells, but exhibited bright fluorescence in TK1-positive MCF-7 cells (Fig. 2c,d). Control experiments using agRNA–3×SRB2 alone or a scrambled agRNA–3×SRB2 in MCF-7 cells also displayed very low fluorescence. Flow cytometry showed a consistent, population-based result for the TK1-responsive system (Fig. 2e, Supplementary Fig. 4). qRT-PCR analysis showed that the bright fluorescence was associated with high abundance of agRNA–3×SRB2 (Supplementary Fig. 4). Together, these results demonstrated that both dCas9 and target RNAs were essential for activating the fluorescence of SRB2, verifying the protection of agRNA–3×SRB2 by dCas9 in specific response to TK1 mRNA.

The system was further exploited for detecting TK1 mRNA in different cells. We found that the fluorescence intensities varied in cell lines with varying TK1 expressions²⁹ (Fig. 2f). TK1-positive cell lines, MCF-7, HeLa and HepG2, exhibited intense fluorescence, whereas TK1-negative cell lines, MCF-10A and BRL, showed negligible fluorescence, with remarkable fluorescence signal-to-background ratios for the positive to negative cells. Ratiometric images of SRB2 fluorescence to the transfection control BFP fluorescence (R/B) showed distinct color shifts across these cells. The average R/B ratios were consistent with the relative TK1 expressions as determined by qRT-PCR (Fig. 2g), which was in good agreement with literature reports²⁹. Our system was also applied to TK1 imaging in MCF-7 cells treated with an up-regulating drug β-estradiol or a down-regulating drug tamoxifen^29,31. The cells stimulated with β-estradiol delivered stronger fluorescence, whereas those treated with tamoxifen displayed much weaker fluorescence (Supplementary Fig. 5). The average R/B ratios were dynamically correlated to the relative TK1 expressions determined by qRT-PCR (Fig. 2h). Collectively, these results demonstrated that the LUSTER system exhibited clear potential for quantitatively imaging of TK1 mRNA in live cells.

Subcellular targeting and multicolor imaging. We next investigated whether the LUSTER system could allow targeting RNAs of varying subcellular localization without altering their localization. We designed an exogenous RNA sequence fused with a specific subcellular-localization tag, a 5S ribosomal RNA in cytosol¹², an ER target mRNA-CytERM (cytoplasmic end of an endoplasmic reticulum signal-anchor membrane protein)³², or a 7SK RNA in nucleus¹² (Supplementary Table 3). FISH and organelle staining verified that the fusion RNAs with 5S ribosomal RNA, CytERM and 7SK RNA tags displayed localization in cytosol, ER and nucleus, respectively (Extended Data Fig. 4a-c). Targeting of the exogenous RNA sequences using the LUSTER system gave bright fluorescence responses colocalized with FISH signals for the corresponding fusion RNAs (Pearson’s correlation coefficients (PCCs) values > 0.85). This result validated that our system allowed imaging of RNAs in the nucleus and cytoplasm without altering their subcellular localization.

Next, we explored the potential of our system for multicolor imaging and its broad applicability to other light-up aptamers. We substituted 3×Broccoli⁶, 3×Mango⁷ or 3×Pepper¹² for the 3×SRB2 motif in the TK1 mRNA-responsive agRNA–3×SRB2 (Supplementary Table 3). These light-up aptamer embedded agRNAs showed strong fluorescence in TK1-positive MCF-7 cells in the presence of cognate dyes, whereas the fluorescence was low in TK1-negative MCF-10A cells and MCF-7 cells expressing only these agRNAs (Fig. 2i and Supplementary Fig. 2). The signal-to-background ratios for MCF-7 to MCF-10A cells were 5.6-, 6.1-, and 10.4-fold, respectively, for Broccoli, Mango and Pepper (Fig. 2j). This result suggested that our system could be universally applicable to varying light-up RNA aptamers with high contrast for mRNA imaging. Though our system was demonstrated for only three spectrally distinct light-up aptamers (green for Broccoli or Mango, orange for SRB2 and near infrared for Pepper), it had the potential to combine other RNA aptamers and further expand the color palette for multicolor RNA imaging.

Single-molecule RNA imaging. The key to our LUSTER system was the RNA-responsive agRNA with embedded aptamers. We wondered whether a larger array of embedded aptamers could afford signal amplification for RNA imaging. To this end, we designed a set of TK1-responsive agRNAs embedded with SRB2 arrays, agRNA–n×SRB2 (n = 3, 6, 12, and 24). A CMV promoter was used for the expression of these agRNAs to avoid the transcription length limit of U6 promoter, and two hammerhead ribozymes (HHRs) were inserted to release the agRNA sequence³³ (Supplementary Fig. 6 and Supplementary Table 3), as validated by RT-qPCR using the agRNA–3×SRB2 design. Microscopic images and flow cytometry showed that MCF-7 cells with dCas9 co-expression displayed higher fluorescence with increasing SRB2 repeats, and signal-to-background ratios over 26.1-fold and 9.0-fold were achieved for 24×SRB2 and 3×SRB2, respectively (Extended Data Fig. 5). This result suggested that our system could exploit a larger array of embedded light-up aptamers for signal amplified imaging.

We further explored whether this signal amplified system could be used for single-molecule RNA imaging. The implementation of single molecule imaging relies on the affinity and photophysical property of the cognate dye toward its RNA aptamer, and single molecule imaging of RNAs have recently been addressed using genetically inserted arrays of light-up RNA aptamers^13,14,16. It was reported that the cognate dye (TMR-DN) of SRB2 had a desirable affinity (~ 36 nM) and photostability, suggesting its potential for RNA image at single molecule resolution⁹. A plasmid was constructed using a miniCMV promoter to express a low-abundance exogenous RNA target with a 24×MS2 tag and a CMV promoter to express the agRNA–24×SRB2 (Fig. 3a,b and Supplementary Table 3). Intense fluorescence puncta from SRB2 were obtained in MCF-7 cells transfected with the exogenous RNAs, whereas negligible fluorescence appeared in control cells without exogenous RNAs (Fig. 3c). To verify whether the fluorescence puncta were specifically attributed to single exogenous RNA molecules, we utilized the MS2–MCP system as a reference method³². We validated that the MS2–MCP system specifically delivered cytosol-localized GFP foci for the exogenous RNA with 24×MS2 tag (Supplementary Fig. 7a). Co-expression of the MS2-MCP and LUSTER systems showed desirable colocalization between the GFP foci and SRB2 puncta on different Z-slices with PCC values over 0.71 (Fig. 3d and Supplementary Fig. 7b). Notably, the SRB2 puncta exhibited a much higher signal-to-background ratio (~ 25.2-fold) than the GFP foci (~ 15.0-fold) (Fig. 3e), highlighting the contrast improvement advantage for our LUSTER system. A closer interrogation revealed that the intensities of GFP foci and SRB2 puncta both followed a single-mode Gaussian distribution (Fig. 3f), consistent with previous reports for fluorescent signals representing single mRNA molecules³⁴. Besides, the SRB2 and GFP puncta exhibited a similar single-mode size distribution (Fig. 3g), recapitulating the single-molecule attribute for these puncta.

Single-molecule fluorescent in situ hybridization (smFISH) was further utilized for validation of our LUSTER system. We observed that the Cy5 fluorescence puncta from smFISH probes exhibited high colocalization with those from SRB2 fluorescence (Fig. 3h and Extended Data Fig. 6a,b). With reference to the smFISH foci, colocalization statistical analysis for Z-sliced images gave an overall labeling efficiency of 74 ± 7%. A further smFISH assay for cells expressing the MS2–MCP system also showed colocalization of the Cy5 fluorescence puncta with the GFP foci with an overall labeling efficiency of 69 ± 8% (Extended Data Fig. 6c,d), which was close to literature data for the MS2–MCP system²¹. Together, these results demontrated that our LUSTER system afforded high efficiency for single molecule imaging.

Next, we further investigated the utility of our system for single-molecule detection of an endogenous low-abundance mRNA of MUC4²¹. We designed an agRNA–24×SRB2 responsive to a non-repetitive region of MUC4 mRNA (Fig. 3i and Supplementary Table 3). As expected, MUC4-positive MCF-7 cells showed bright SRB2 fluorescence puncta, and a control using small interfering RNAs (siRNAs) for MUC4 knock-down testified the specificity of our system (Extended Data Fig. 7a,b). Intensity and size analysis revealed that these puncta were ascribed to single molecules (Extended Data Fig. 7c). A smFISH assay showed clear colocalization between the SRB2 puncta and the smFISH probes with a 73 ± 6% overall labeling efficiency (Fig. 3i and Extended Data Fig. 7d,e). Additionally, qRT-PCR and western blotting experiments verified that our system had little effect on the expression and translation of MUC4 mRNA (Extended Data Fig. 7f-h). Collectively, these results validated that the LUSTER system was capable of single-molecule imaging of low-abundance mRNA with high specificity and labeling efficiency.

Imaging of RNA localization and dynamics. We investigated the potential of our system for dynamic imaging of β-actin mRNA translocation using a designed agRNA–24×SRB2 responsive to β-actin (Supplementary Table 3). Co-transfection of agRNA–24×SRB2 with dCas9 in MCF-7 cells displayed clear SRB2 fluorescence puncta, and a control experiment with β-actin depleted by siRNAs confirmed the specificity of our system (Supplementary Fig. 8a,b). qRT-PCR and western blotting analysis confirmed that our system did not affect the abundance and translation of β-actin mRNA (Supplementary Fig. 8c-e). A smFISH assay confirmed the specificity of our system for β-actin labeling (Fig. 4a). The dynamic translocation of β-actin to stress granules (SGs) was further interrogated with reference to a fusion protein of GFP with G3BP1, a SG marker¹⁵. After MCF-7 cells treated with arsenite for oxidation stress induction³⁵, the SRB2 fluorescence puncta became aggregated into larger foci and colocalized with the GFP puncta (Fig. 4b). With GFP foci as the indicator, the labeling efficiency of SGs by SRB2 fluorescence was 79 ± 7%. In addition, the SRB2 fluorescence foci showed a remarkably broadened distribution in intensity and size after arsenite treatment (Fig. 4c,d), implying that individual SGs were composed of multiple copies of β-actin mRNA. It was noteworthy that we occasionally found aggregated GFP foci in transfected cells before arsenite stimulation (Supplementary Fig. 9), consistent with the literature result that SGs could form due to transfection¹⁵. A time-lapse study showed that remarkable assembly of SGs appeared within 20 min after induction with a synchronous merge of SRB2 puncta, and the assembly process was completed in ~ 90 min as the size and intensity of SGs became stabilized (Extended Data Fig. 8a,b). Together, these results demonstrated the capability of our system for dynamic imaging of mRNA translocation in live cells.

We next exploited our system for detecting the dynamics of long noncoding RNA (lncRNA) in nucleus using a model target NEAT1 (NEAT1-2), a structural lncRNA component localized in paraspeckles³⁶. Co-transfection of an agRNA–24×SRB2 responsive to NEAT1 with dCas9 in MCF-7 cells delivered bright SRB2 fluorescence foci with clear nuclear localization (Extended Data Fig. 9a,b). Depletion of NEAT1 using siRNAs resulted in a remarkable decrease in SRB2 puncta and GFP foci, confirming the specificity of SRB2 puncta to NEAT1 and the essential role of NEAT1 in paraspeckle formation³⁶. The SRB2 foci were colocalized with the fluorescence puncta from a GFP fusion with a paraspeckle marker NONO³⁷ (Extended Data Fig. 9c,d). Z-stacked images showed 20 ± 3 GFP-labeled paraspeckles in single cells, consistent with the literature result³⁶, and 85 ± 6% of paraspeckles were labeled by SRB2 foci (Extended Data Fig. 9e). A smFISH assay validated our system in specific detection of NEAT1 (Fig. 4e). To shed light on the dynamics of NEAT1 in paraspeckle organization, we treated the cells with stimulants for NEAT1 regulation. For MCF-7 cells stimulated with arsenite to upregulate NEAT1 via oxidative stress induction³⁸ (Extended Data Fig. 10a,b), we observed an increased number of highly colocalized fluorescence puncta for SRB2 and GFP (Fig. 4f and Extended Data Fig. 10c,e). The paraspeckles exhibited an enlarged size and enhanced SRB2 fluorescence, indicating that the expanded paraspeckles needed more NEAT1 structures (Fig. 4g,h). On the other hand, after MCF-7 cells were stimulated with 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB), an RNA polymerase II inhibitor for NEAT1 downregulation³⁹ (Extended Data Fig. 10a,b), the paraspeckles became disintegrated as indicated by decreased size and number of the colocalized fluorescence puncta for SRB2 and GFP, accompanied with decreased fluorescence intensities for SRB2 (Fig. 4g,h and Extended Data Fig. 10d,e). This result suggested the function of NEAT1 in facilitating the assembly of paraspeckles, consistent with previous findings³⁶. Collectively, these results demonstrated the potential of our system for imaging RNA localization and dynamics in the nucleus.

RNA induced transcriptional activation. The LUSTER system could be further extended for RNA-inducible genome engineering by appending a DNA-targeting spacer sequence to agRNA. To re-direct our system for transcriptional activation, we deployed a nuclear-localized dCas9–VPR fusion⁴⁰ and utilized the agRNA–3×SRB2 responsive to TK1 mRNA for demonstration (Fig. 4i). An iRFP reporter plasmid with eight spacer-binding target sites upstream to a miniCMV promoter was designed for direct visualization of transcriptional activation (Fig. 4j). Upon transfection with dCas9-VPR, agRNA–3×SRB2 and the reporter plasmid, TK1-positive MCF-7 cells displayed bright SRB2 fluorescence accompanied with strong iRFP fluorescence, whereas TK1-negative MCF-10A cells delivered very low SRB2 fluorescence with a weak iRFP signal (Fig. 4k). Further controls using correctly-folded gRNA–3×SRB2 in TK1-negative MCF-10A cells and an agRNA with 13-bp scrambled complementary regions in TK1-positive MCF-7 cells confirmed TK1-induced dCas9-stabilized agRNA–3×SRB2 and transcription activation by the LUSTER system (Supplementary Fig. 10a). Time-lapse fluorescence images revealed precedent fluorogenic responses for SRB2 and subsequent expression of iRFP (Fig. 4l and Supplementary Fig. 10b). Together, these results demonstrated that our LUSTER system enabled RNA-induced transcriptional activation, highlighting its potential for cell-specific genome engineering applications by combining recently developed Cas-derived base modifying enzymes.

We have developed a programmable LUSTER system that enables fluorogenic imaging of RNAs in live mammalian cells. Our system relies on the recognition of Cas9/dCas9 toward correctly folded gRNA, stabilizing gRNA against degradation. By engineering an abrogated gRNA with conformation restored by target RNAs, our system provides a new RNA-responsive tool to control CRISPR/dCas9 activity. We exploited the stabilization property of small light-up RNA aptamers in mammalian cells to design light-up aptamer embedded agRNAs, creating an activatable system for RNA imaging and dynamic tracking.

Compared with the pilot dCas9 or dCas13 based RNA recognition approach, our system offers several advantages, including fluorogenicity and signal amplifiability without the need of the PAMmer oligonucleotide and related delivery treatments. Compared with the versatile light-up RNA strategy, our system allows direct imaging of endogenous RNA without need of complicated genetic manipulations to insert the aptamer tags, thus opening the possibility for theranostic applications. The improvements are also beneficial for imaging or flow-cytometry detection as well as high-contrast tracking for RNAs and even for low-abundance targets.

We have demonstrated that our system is generally applicable to quantitative detection and dynamic imaging of diverse RNA targets. It allows intensity-based fluorescence monitoring of TK1 mRNA expressions and their dynamic changes in different cells. By utilizing larger arrays of inserted light-up aptamers, our system offers an elegant design for signal amplification. We have shown the ability of our system with 24×SRB2 aptamers for single-molecule RNA detection. This single-molecule RNA imaging strategy confers higher signal-to-background ratios with consistent labeling efficiency as the standard MS2-MCP system. Our system is demonstrated to have negligible interference with subcellular localizations and physiological functions of target RNAs. Moreover, we have demonstrated the ability of our system for mapping β-actin mRNA translocation in SGs and lncRNA NEAT1 dynamics in paraspeckles. Finally, our system has been extended toward an RNA-inducible transcriptional activation tool for genome engineering.

Our system is generally applicable to different light-up aptamers. Our results have shown examples for two green fluorescent aptamers including Broccoli or Mango, one orange fluorescent aptamer SRB2, and one NIR fluorescent aptamer Pepper. By designing agRNAs specific to three different RNA targets, our system can be extended for triplexed imaging. With the ever-growing light-up aptamer arsenal, we expect that our system could further expand its color palette and enhance the capability for multiplexed RNA imaging.

The major limitation of our system lies in the affinity of dCas9 toward gRNA, which is crucial for stability of the dCas9–gRNA complex in mammalian cells. We demonstrate that a fully extended gRNA has higher affinity toward dCas9 and increases the stability of the complex and thus affords high detection efficiency. Further optimization of gRNA structure, evolution of dCas9 mutants and search of CRISPR/dCas9, dCas12, and even RNA-targeting dCas13 homologs for enhanced affinity has the potential to achieve better sensitivity.

Overall, our LUSTER system offers as a new RNA imaging tool featuring several favorable properties such as fluorogenicity, signal amplifiability, trackability and genome targetability. It is programmable, modular and generally applicable to different RNAs, highlighting its great potential for RNA studies and RNA-induced genome engineering.

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Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements; details of author contributions and competing interests; and statements of data and code availability are available.

Acknowledgements

This work was supported by National Key Research Program (2019YFA0905800, 2018YFA0905800) and NSFC Programs (22090050, 21991084, 21904034).

Author contributions

J.H.J. conceived and designed the project. F.Z. performed majority of the experiments. F.L. performed in vitro cleavage and gRNA binding assays. F.Z., F.W., X.C. and J.H.J. conducted data analysis and interpretation. J.H.J. wrote the manuscript and supervised the project. All authors read and approved the final manuscript.

Competing interests

The authors declare no competing interests.

Additional information

Extended data is available for this paper.

Supplementary information is available for this paper

Correspondence and requests for materials should be addressed to J. H. Jiang.

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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). C_t values for target RNAs and GAPDH were determined and ΔC_t values were obtained by subtracting the C_t 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.

Data availability

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.

References

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).

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A programmable CRISPR-based light-up RNA stabilizer for fluorogenic RNA imaging

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