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

doi:10.21203/rs.3.rs-1435078/v3

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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/v3

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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 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 and multiplexed RNA imaging, cytosolic or nuclear RNA localization imaging, and RNA-induced orthogonal 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 live cell imaging of RNAs are more challenging and largely rely on two strategies: multimeric protein-binding RNA tags that recruit fluorescent protein fusions^1-4, and fluorescence self-quenched hybridization probes^5-8. The multimeric RNA tag system generally involves 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 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^9-16. Genetic incorporation of fluorogenic aptamer tags into target RNAs allows direct visualization of their expression and localization^17-21. Significant improvements using RNA aptamer stabilized fluorescent proteins have provided a completely genetically encodable tool for fluorogenic live cell imaging of target RNAs²². Unfortunately, most fluorogenic aptamer systems require genetic insertion of aptamer motifs into target RNAs, precluding non-invasive imaging for endogenous RNAs. Additionally, these systems usually exhibit limited brightness and photostability in mammalian cells, compromising the ability for single-molecule RNA detection. Another important advance is the clustered regularly-interspaced short palindromic repeats (CRISPR) system for RNA targeting and imaging^23-27. A nuclease-deficient Cas9 (dCas9) system with fluorescent protein fusion, when combined with a synthetic PAMmer oligonucleotide, has been repurposed for programmable targeting and visualization of high-abundance endogenous RNAs^23,24. More recently, the CRISPR/dCas13 system has demonstrated as a more versatile tool for labeling and tracking of RNAs^25,26. 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 fluorescent proteins. 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 a fully extended gRNA and stabilize it against degradation in vitro or in mammalian cells. By optimizing 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 demonstrated for mRNA detection, cytosolic mRNA translocation imaging, nuclear lncRNA localization, single-molecule and multiplexed RNA imaging, and RNA-induced orthogonal transcriptional activation in mammalian cells. We expect that the LUSTER system can provide a broadly useful, programmable platform for RNA imaging and RNA-activated genome engineering.

Cas9 stabilizes gRNA with embedded light-up RNA. In CRISPR system, Cas9/dCas9 protein could bind gRNA and mediate recognition of target DNA, driving significant conformational changes within Cas9/dCas9 to form a binary or ternary complex^28,29. In cellular environments, gRNAs are highly unstable and susceptible to degrade^30,31, but exhibit higher stability within the Cas9-gRNA-DNA ternary complex^26,32. However, the stability of gRNAs in the Cas9/dCas9-gRNA binary complex and its utility as molecular tools have been largely unexplored. We envisioned that a structural optimized gRNA could enhance its recognition to Cas9/dCas9 and be stabilized against degradation in live cells. Motivated by this hypothesis, we considered developing an RNA imaging tool by combining dCas9-stabilized gRNAs with embedded light-up aptamers. As most of light-up aptamers are short RNAs that exhibit limited stability and rapid degradation in mammalian cells, these aptamers can act as a conditionally fluorescent reporter if they are stabilized in the dCas9-protected gRNAs scaffold. In this way, fluorescence can only be selectively activated by the formation of dCas9-gRNA complex, affording fluorogenic signals for the stabilized aptamer-embedded gRNAs, or target RNAs that modulate the binding of gRNAs to dCas9. This concept enabled developing a programmable CRISPR-based light-up RNA Stabilizer (LUSTER) system for RNA imaging.

The key to the LUSTER system is the design of structural optimized gRNAs that have improved affinity to dCas9. Previous studies showed that a structurally extended gRNA (egRNA) enhanced its interaction with dCas9³³, enabling a CRISPR tool for imaging the chromatin conformation. Therefore, we designed three gRNAs with varying structural extensions (Fig. 1a and Supplementary Table 1), the short-version gRNA (sgRNA)³⁴, the egRNA³³ and a fully extended gRNA (fgRNA). These gRNAs were all protected from ribonuclease A (RNase A) degradation in the presence of Cas9, with fgRNA displaying the strongest protection (Extended Data Fig. 1a,b). We ascribed the Cas9-mediated protection of gRNAs to the formation of stable binary Cas9–gRNA complexes (Extended Data Fig. 1c), with the best association achieved for fgRNA. The Cas9–fgRNA complex also exhibited a higher rate in target DNA cleavage than the sgRNA and egRNA counterparts (Extended Data Fig. 1d).

To examine dCas9-mediated stabilization of gRNAs in mammalian cells, we engineered three gRNAs embedded with a light-up aptamer array, 3´SRB2, an RNA aptamer that can specifically bind and activate the fluorescence of tetramethylrhodamine-dinitroaniline (TMR-DN)¹³, in the tetra-loop region³⁵ (Extended Data Fig. 2a, Supplementary Table 1, and Supplementary Fig. 1) and a cytosol-localized dCas9 (Supplementary Fig. 2). The embedded light-up aptamers showed negligible effect on Cas9–gRNA complex in target DNA cleavage (Supplementary Fig. 3), consistent with literature reports that the tetra-loop region of gRNA was tolerant to sequence insertion and extensions of the tetraloop did not affect Cas9–gRNA activity³⁵. Confocal images and flow cytometry showed that these aptamer-embedded gRNAs were unstable with negligible fluorescence (Fig. 1b,c, and Extended Data Fig. 2b,c), consistent with previous findings that gRNAs were rapidly degraded in mammalian cells^26,^31,32. In contrast, dCas9 co-expression displayed bright and homogenous cytosolic fluorescence, suggesting dCas9-mediated stability of these gRNAs. qRT-PCR confirmed the stabilization of these gRNAs under dCas9 co-expression, with the best stability for fgRNA (Extended Data Fig. 2d). RNA transcription inhibition assay with actinomycin D (Act D) showed prolonged fluorescence for gRNAs under dCas9 co-expression, with the most persistent signal obtained for fgRNA (Extended Data Fig. 2e). Together, these results revealed that dCas9 could protect gRNAs in mammalian cells, and fgRNA exhibited the best stability due to its improved association to dCas9.

Design of RNA-responsive gRNA. We explored to develop the RNA imaging system based on dCas9-stabilized gRNA with embedded light-up aptamers by using fgRNA, considering its improved association with dCas9. This system relied on the design of an abrogated gRNA (agRNA) that was misfolded and incapable of binding dCas9 protein due to two inserted complementary sequences, but restored its dCas9-binding conformation via target RNA mediated strand displacement. Structural studies of Cas9–gRNA complex revealed that the repeat:anti-repeat duplex and stem-loop 1 had extensive interactions with Cas9, whereas the three regions (tetra-loop, stem-loop 2 and 3’ terminus) were solvent-exposed and tolerant to sequence variations^28,29. Thus, we tested three designs of agRNAs by inserting complementary sequences in two of the three candidate regions with varying length (11-14 base pairs) (Fig. 1d).

We first designed and optimized agRNAs (Supplementary Table 2) for a model target TK1, an mRNA overexpressed in cancer cells³⁶. In vitro DNA cleavage assay revealed that four agRNAs, with 13- or 14-nucleotide complementary sequence 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. 1e). Previous reports showed that stem-loop 1 was essential in forming the active Cas9–gRNA complex, and stem-loop 2 supported the stable complex formation and improved the in vivo activity^28,37. Our result further confirmed the critical role of stem-loop 2 in supporting the activity of Cas9–gRNA complex. As a matter of fact, the 13- or 14-nucleotide complementary sequence occupied the entire loop of stem-loop 2 in the agRNA, and the formation of rigid duplex between the complementary regions would totally disrupt stem-loop 2, thus substantially decreasing its association with Cas9 and abolishing the cleavage activity.

We next investigated the responsiveness of these four agRNAs to TK1 mRNA. Two agRNAs with 13 base-pair complementation between stem-loop 2 and the tetra-loop or 3’ terminus exhibited high cleavage activity upon mRNA hybridization (Fig. 1f). 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 gRNA with potentially improved cellular stability³⁸. A further assay showed that this agRNA exhibited negligible binding to Cas9, and hybridization with TK1 mRNA restored its binding ability (Extended Data Fig. 3a). Moreover, TK1 hybridization stabilized the agRNA from RNase A-mediated degradation in the presence of Cas9 (Extended Data Fig. 3b). Additionally, the restored Cas9 activity was dynamically dependent upon TK1 concentration (Extended Data Fig. 3c) and specific to TK1–agRNA hybridization (Extended Data Fig. 3d). Collectively, these results suggested that the optimized design of agRNA abrogated its binding to Cas9/dCas9, but restored the correct gRNA conformation in response to target RNA, enabling RNA-responsive control of the CRISPR system.

Live cell imaging of TK1 mRNA. The RNA-responsive gRNA design was exploited to develop the LUSTER imaging system using two plasmid constructs: one for the agRNA embedded with 3´SRB2 (agRNA-3´SRB2) under a U6 promoter and the other for the cytosol-localized dCas9 under an EF1a promoter (Fig. 2a,b, Supplementary Fig. 4 and Supplementary Table 3). 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 with a 10.6-fold positive-to-negative ratio (Fig. 2c,d). Control experiments using agRNA-3´SRB2 alone or a scrambled agRNA-3´SRB2 in MCF-7 cells displayed very low fluorescence. Flow cytometry showed a consistent, population-based result for the TK1-responsive system (Fig. 2e and Supplementary Fig. 5a,b). qRT-PCR testified that the bright fluorescence was associated with high abundance of agRNA-3´SRB2 (Supplementary Fig. 5c,d). These results verified that the stabilization of agRNA-3´SRB2 by dCas9 was highly specific to TK1 mRNA. Additionally, qRT-PCR and western blotting showed that the LUSTER system had negligible effect on the expressions of TK1 mRNA and protein (Supplementary Fig. 6).

The system was further used to detect TK1 mRNA in different cells³⁶. TK1-positive cell lines exhibited intense fluorescence, affording remarkable positive-to-negative ratios as compared to TK1-negative cell lines (Fig. 2f). The SRB2 fluorescence was in good correlation to the TK1 copy number as determined by qRT-PCR (Fig. 2g and Supplementary Fig. 7). Moreover, the fluorescence substantially increased in MCF-7 cells stimulated with TK1 up-regulating drug β-estradiol³⁶, while decreased remarkably upon treatment with down-regulating drug tamoxifen³⁹, with the average SRB2 fluorescence dynamically correlated to the TK1 copy number (Fig. 2h, Supplementary Figs. 7 and 8). Collectively, these results demonstrated the ability of the LUSTER system for quantitatively imaging of TK1 mRNA in live cells.

Subcellular targeting and multiplexed imaging. We investigated whether the LUSTER system could allow targeting RNAs of varying subcellular localization without altering their localization. We designed an agRNA-targeting sequence fused with a specific localization tag, 5S ribosomal RNA in cytosol¹⁴, ER-targeting mRNA encoding CytERM (cytoplasmic end of an endoplasmic reticulum signal-anchor membrane protein)²², or 7SK RNA in nucleus¹⁴ (Supplementary Table 3). Organelle staining and fluorescence in situ hybridization (FISH) verified that the fusion RNAs with 5S ribosomal RNA, CytERM and 7SK RNA tags, respectively, displayed localization in cytosol, ER and nucleus (Extended Data Fig. 4). Targeting of these RNA sequences using the LUSTER system gave bright fluorescence colocalized with FISH probes for the corresponding fusion RNAs (Pearson’s correlation coefficients, PCCs ³ 0.86). This result validated that our system allowed imaging of RNAs in the nucleus and cytosol without altering their subcellular localization.

Next, we explored the applicability of our system to other light-up aptamers for multicolor RNA imaging. We substituted 3´Broccoli¹⁰, 3´Mango¹¹ or 3´Pepper¹⁴ for 3´SRB2 in the TK1-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 (Supplementary Fig. 1), in stark contrast with the negligible fluorescence in TK1-negative cells or MCF-7 cells without dCas9 expression (Fig. 2i). The positive-to-negative 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.

We further investigated the possibility of our LUSTER system for multiplexed imaging by combining the TK1-responsive agRNA-3´SRB2 with other two designed agRNAs, agRNA-3´Mango and agRNA-3´Pepper, responding to c-myc and GalNAc-T mRNAs, respectively (Supplementary Table 3). Co-transfection of three agRNAs and dCas9 in MCF-7 and HepG2 cells (c-myc, TK1 and GalNAc-T positive) delivered bright fluorescence for Mango, SRB2 and Pepper, in contrast to the negligible fluorescence in MCF-10A cells with negative expression of the three targets (Extended Data Fig. 5a-c). The orthogonality was examined via small interfering RNA (siRNA) mediated depletion of target mRNA (Supplementary Table 3), and substantially decreased fluorescence was obtained in MCF-7 cells for SRB2, Mango and Pepper, respectively, only when TK1, c-myc, TK1 and GalNAc-T were depleted (Extended Data Fig. 5d-f). Together, these results validated the potential of the LUSTER system for multi-color and multiplex imaging of RNAs in live cells.

Single-molecule RNA imaging. We wondered whether a larger array of embedded aptamers could afford signal amplification for RNA imaging. 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 expressing these agRNAs to avoid the transcription length limit of U6 promoter, and two hammerhead ribozymes⁴⁰ (HHRs) were inserted to release the agRNAs (Supplementary Fig. 9a,b and Supplementary Table 3). The release of agRNAs by HHRs was validated by qRT-PCR using the agRNA-3´SRB2 design (Supplementary Fig. 9c,d). Similar to agRNA-3´SRB2, agRNA-24´SRB2 also had negligible effect on the cleavage activity of Cas9 (Supplementary Fig. 10). Microscopic images and flow cytometry showed that MCF-7 cells with dCas9 co-expression displayed higher fluorescence with increasing SRB2 repeats, and positive-to-negative ratios of 26.1-fold and 9.0-fold were achieved for 24´SRB2 and 3´SRB2, respectively (Extended Data Fig. 6). 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. A plasmid was constructed using a CMV promoter to express the agRNA-24´SRB2 as well as a miniCMV promoter to express a low-abundance exogenous RNA containing a 24´MS2 tag and an agRNA-targeting sequence (Fig. 3a,b and Supplementary Table 3). The implementation of single molecule imaging relied on the aptamer-binding affinity and photophysical properties of the cognate dye, and single molecule imaging of RNAs had recently been achieved using light-up aptamers^18-21. The cognate dye (TMR-DN) of SRB2 was reported to have a desirable affinity (~36 nM for K_d) and photostability^13,20, suggesting its potential for RNA image at single molecule resolution. To verify the ability of our system for single-molecule RNA imaging, we utilized the MS2–MCP system¹ as the reference. As anticipated, the MS2–MCP and LUSTER systems both delivered intense fluorescence puncta in MCF-7 cells transfected with the exogenous RNAs, exhibiting desirable cytosolic colocalization with a PCC of 0.83 (Fig. 3c). Moreover, the fluorescence puncta from the MCP-MS2 and our system were both validated to be specific to the MS2 tag and the agRNA-targeting sequence, respectively (Supplementary Fig. 11). A closer interrogation of the fluorescence intensity and size distribution revealed a single-mode population for the GFP or SRB2 foci with a diffraction-limited size (Fig. 3d,e), recapitulating the single-molecule attribute for these puncta. Notably, the SRB2 puncta exhibited a much higher signal-to-background ratio (19.1-fold) than the GFP foci (14.2-fold) (Fig. 3f). A further comparison with the MCP–MS2 system using a fluorescence protein (mCherry) with the same detection channel as SRB2 also showed a higher signal-to-background ratio (18.9-fold versus 15.5-fold) (Supplementary Fig. 12), highlighting the contrast advantage for our LUSTER system.

Single-molecule FISH (smFISH) was further utilized for validation of our LUSTER system (Extended Data Fig. 7a). Likewise, the Cy5 fluorescence puncta from smFISH probes exhibited high colocalization with those from SRB2 with a PCC of 0.79 (Extended Data Fig. 7b). Control experiments validated that the fluorescence puncta for SRB2 and Cy5 were specific to the target RNA with no crosstalk between the two detection channels (Supplementary Fig. 13). A further analysis revealed diffraction-limited sizes for the SRB2 or Cy5 puncta with a single-mode distribution for the fluorescence intensities and sizes (Extended Data Fig. 7c). The ability of LUSTER system for single molecule imaging was further demonstrated using a miniP promoter to express the RNA target in a lower abundance (Supplementary Fig. 14a,b). Colocalized and diffraction-limited puncta were obtained for SRB2 and smFISH probes (PCC 0.80) (Supplementary Fig. 14c-e).

We further investigated the utility of our system for single-molecule detection of an endogenous mRNA of MUC4²⁵ by targeting a non-repetitive region of MUC4 (Supplementary Table 3). As expected, MUC4-positive MCF-7 cells displayed bright SRB2 puncta of a diffraction-limited size (Fig. 3g-i). A control using MUC4-depleting siRNAs testified the specificity of these SRB2 puncta. Moreover, the SRB2 puncta showed clear colocalization (PCCs ³ 0.75) with the smFISH probes in all the Z-slices (Fig. 3j and Supplementary Fig. 15). Additionally, qRT-PCR and western blotting verified that our system had little effect on the expressions of MUC4 mRNA and protein (Supplementary Fig. 16).

We further investigated the LUSTER system for tracking single-molecule translocation of an ER-targeted mRNA. Confocal images showed obvious SRB2 puncta with diffraction-limited size for the mRNA (Supplementary Fig. 17). Time-lapse imaging revealed low mobility for the SRB2 puncta (Fig. 3k,m and Supplementary Video 1), consistent with the restricted mobility of the ER-targeted mRNA during translation²². The mobility of SRB2 puncta increased remarkably upon treatment with puromycin, which dissociated the ER-targeted mRNAs²² (Fig. 3l,m and Supplementary Video 2). Quantitative calculations revealed much smaller diffusion coefficients for the ER-targeted mRNAs as compared to the dissociated mRNAs (Fig. 3n). Collectively, these results validated that the LUSTER system was capable of single-molecule RNA imaging with high specificity and contrast.

Imaging of RNA localization and dynamics. We investigated the potential of our system for dynamic imaging of β-actin mRNA translocation using a β-actin responsive agRNA-24´SRB2 (Supplementary Table 3). Co-transfection of agRNA-24´SRB2 with dCas9 displayed clear SRB2 puncta, and a control with β-actin-depleting siRNAs confirmed that the SRB2 puncta were specific to β-actin mRNAs (Supplementary Fig. 18a,b). A smFISH assay further validated the specificity of our system for β-actin labeling (Fig. 4a). qRT-PCR and western blotting testified that our system did not affect the abundance and translation of β-actin (Supplementary Fig. 18c-e). To interrogate the dynamic translocation of β-actin to stress granules (SGs), we used GFP–G3BP1, an SG marker¹⁷, as the reference. After stress induction with arsenite¹⁷, the SRB2 puncta became aggregated into larger foci and colocalized to the GFP puncta with a PCC of 0.82 (Fig. 4b). A remarkably broadened distribution in intensity and size for the SRB2 foci indicated the assembly of multiple copies of β-actin mRNA in the SGs (Fig. 4c,d). A time-lapse study showed that substantial assembly of SGs appeared within 20 min after induction with a synchronous merge of SRB2 puncta, and the assembly process was completed in ~120 min (Extended Data Fig. 8). 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 a long noncoding RNA (lncRNA) of NEAT1-2, a structural component of paraspeckles⁴¹. Co-transfection of an agRNA-24´SRB2 responsive to NEAT1 with dCas9 delivered bright SRB2 foci colocalized with smFISH probes, confirming that our system was specific in NEAT1 detection (Fig. 4e). The SRB2 foci were also colocalized with a paraspeckle marker GFP–NONO⁴² (Extended Data Fig. 9a,b). A NEAT1-depleted control displayed a remarkable decrease in SRB2 puncta and GFP foci, validating the specificity of SRB2 puncta to NEAT1 and the essential role of NEAT1 in paraspeckle formation⁴¹. A closer interrogation along the Z-stacked images testified the high colocalization of SRB2 puncta to GFP foci with PCCs over 0.83 (Extended Data Fig. 9c,d). The GFP-labeled paraspeckles was counted up to 20 ± 3 per cell, consistent with the literature result⁴¹ (Extended Data Fig. 9e). The dynamics of NEAT1 in paraspeckle organization was further investigated. Upon NEAT1 upregulation via arsenite stimulation⁴², we obtained an increased number of highly colocalized SRB2 and GFP puncta (Fig. 4f-h and Extended Data Fig. 10). The enhanced SRB2 fluorescence within the enlarged paraspeckles suggested that the paraspeckle growth included more NEAT1 structures. Upon NEAT1 down-regulation via treatment with an RNA polymerase II inhibitor 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB)⁴³, the paraspeckles became disintegrated into fewer and smaller phases with decreased SRB2 fluorescence. This result suggested the function of NEAT1 in facilitating the assembly of paraspeckles⁴¹. 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 using an agRNA with a DNA-targeting spacer sequence and a nuclear-localized dCas9-VPR fusion⁴⁴. We started from a model system using the TK1-responsive agRNA and an iRFP reporter plasmid with eight spacer-binding target sites upstream to a miniCMV promoter (Fig. 5a,b). As anticipated, TK1-positive MCF-7 cells displayed bright SRB2 fluorescence accompanied with strong iRFP fluorescence, in stark contrast to the TK1-negative MCF-10A cells with negligible SRB2 and iRFP signals (Fig. 5c). A reporter plasmid with only one or two spacer-binding sites also delivered intense iRFP fluorescence (Supplementary Fig. 19). Further controls confirmed the specificity of the LUSTER system in TK1-induced transcription activation (Supplementary Fig. 20a). Time-lapse fluorescence images suggested that it took ~4 h for iRFP expression after stabilization of the TK1-responsive agRNA (Fig. 5d and Supplementary Fig. 20b).

We also exploited the LUSTER system for inducible transcription of endogenous RNAs (Fig. 5e). A β-actin responsive agRNA was designed to activate the transcription of two endogenous mRNAs, ASCL1 and IL1RN, using spacer sequences targeting one or two promoter regions of these two genes. qRT-PCR revealed that both mRNA expressions were significantly elevated and higher expressions were obtained by targeting two promoter regions, in contrast to the negligible expression in the control using a scrambled agRNA not responsive to β-actin (Fig. 5f). The inducible transcription system was further validated using a TK1-responsive agRNA targeting promoter regions of IL2 and IFN-γ, and consistently elevated expressions were achieved for these two genes (Fig. 5g).

The LUSTER system was further investigated for orthogonal transcription activation of IL2 and IFN-γ, respectively, using c-myc and TK1-responsive agRNAs. As expected, activated expression of IL2 was achieved specifically in the c-myc-positive cells with a concentration-dependency upon the inducer c-myc mRNA (Fig. 5h and Extended Data Fig. 5a,d). Likewise, TK1 mRNA concentration-dependent transcription activation of IFN-γ was obtained in the TK1-positive cells (HepG2 and MCF-7) in contrast to the negative cell line (MCF-10A). Moreover, siRNA-mediated depletion of c-myc or TK1 in MCF-7 cells confirmed the orthogonality in transcription activation of IFN-γ and IL2 by the corresponding mRNA inducers. Together, these results demonstrated that our LUSTER system enabled RNA-induced transcriptional activation with high selectivity and orthogonality, highlighting its potential for cell-specific genome engineering applications.

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 advantages such as fluorogenicity or low background, multiplexity and signal amplifiability for contrast improvement. Compared with the versatile light-up RNA strategy, our system allows direct imaging of endogenous RNA without need of genetic insertion of 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 localization and tracking. This single-molecule RNA imaging strategy confers higher signal-to-background ratios than 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. In addition, our system has been extended toward a transcriptional activation tool that enables orthogonal RNA induction for genome engineering.

Our system is generally applicable to different light-up aptamers. Our results have shown examples for four fluorescent aptamers and further demonstrated the capability for triplexed imaging of three RNAs. With the ever-growing light-up aptamer arsenal, we expect that our system could further expand its color palette and enhance the capacity for multiplexed RNA imaging. Our gRNA design provides a general strategy for conditional activation for CRISPR-Cas9 systems because structure switching of gRNAs are highly sequence specific, affording orthogonality, multiplexity and sequence versatility.

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.

Acknowledgements

This work was supported by National Key Research Program (2019YFA0905800, 2018YFA0905800), NSFC Programs (22090050, 21991084, 22274040) and Hunan Provincial Science Fund for Distinguished Young Scholars (2022JJ10004).

Author contributions

J.H.J. conceived and designed the project. Z.F. performed majority of the experiments. F.L. performed in vitro cleavage and gRNA binding assays. Z.F., F.W., X.C. and J.H.J. conducted data analysis and interpretation. J.H.J. and F.L. 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.

Reprints and permissions information is available.

Tutucci, E. et al. An improved MS2 system for accurate reporting of the mRNA life cycle. Nat. Methods 15, 81–89 (2018).
Fu, Y. et al. CRISPR-dCas9 and sgRNA scaffolds enable dual-colour live imaging of satellite sequences and repeat-enriched individual loci. Nat. Commun. 7, 11707 (2016).
Kim, S.H., Vieira, M., Kim, H.J., Kesawat, M.S. & Park, H.Y. MS2 labeling of endogenous beta-actin mRNA does not result in stabilization of degradation intermediates. Mol. Cells 42, 356–362 (2019).
Das, S., Moon, H.C., Singer, R.H. & Park, H.Y. A transgenic mouse for imaging activity-dependent dynamics of endogenous Arc mRNA in live neurons. Sci. Adv. 4, 14 (2018).
Bratu, D.P., Cha, B.J., Mhlanga, M.M., Kramer, F.R. & Tyagi, S. Visualizing the distribution and transport of mRNAs in living cells. Proc. Natl. Acad. Sci. USA 100, 13308–13313 (2003).
Seferos, D.S., Giljohann, D.A., Hill, H.D., Prigodich, A.E. & Mirkin, C.A. Nano-flares: probes for transfection and mRNA detection in living cells. J. Am. Chem. Soc. 129, 15477–15479 (2007).
Turner-Bridger, B. et al. Single-molecule analysis of endogenous β-actin mRNA trafficking reveals a mechanism for compartmentalized mRNA localization in axons. Proc. Natl. Acad. Sci. USA 115, E9697–E9706 (2018).
Santangelo, P.J. et al. Single molecule-sensitive probes for imaging RNA in live cells. Nat. Methods 6, 347–349 (2009).
Paige, J.S., Wu, K.Y. & Jaffrey, S.R. RNA mimics of green fluorescent protein. Science 333, 642–646 (2011).
Filonov, G.S., Moon, J.D., Svensen, N. & Jaffrey, S.R. Broccoli: rapid selection of an RNA mimic of green fluorescent protein by fluorescence-based selection and directed evolution. J. Am. Chem. Soc. 136, 16299–16308 (2014).
Dolgosheina, E.V. et al. RNA mango aptamer-fluorophore: a bright, high-affinity complex for RNA labeling and tracking. ACS Chem. Biol. 9, 2412–2420 (2014).
Sunbul, M. & Jaschke, A. Contact-mediated quenching for RNA imaging in bacteria with a fluorophore-binding aptamer. Angew. Chem. Int. Ed. Engl. 52, 13401–13404 (2013).
Sunbul, M. & Jaschke, A. SRB-2: a promiscuous rainbow aptamer for live-cell RNA imaging. Nucleic Acids Res. 46, e110 (2018).
Chen, X. et al. Visualizing RNA dynamics in live cells with bright and stable fluorescent RNAs. Nat. Biotechnol. 37, 1287–1293 (2019).
Sato, S. et al. Live-cell imaging of endogenous mRNAs with a small molecule. Angew. Chem. Int. Ed. Engl. 54, 1855–1858 (2015).
Yatsuzuka, K. et al. Live-cell imaging of multiple endogenous mRNAs permits the direct observation of RNA granule dynamics. Chem. Commun. 54, 7151–7154 (2018).
Braselmann, E. et al. A multicolor riboswitch-based platform for imaging of RNA in live mammalian cells. Nat. Chem. Biol. 14, 964–971 (2018).
Li, X., Kim, H., Litke, J.L., Wu, J. & Jaffrey, S.R. Fluorophore-promoted RNA folding and Photostability Enables Imaging of Single Broccoli-Tagged mRNAs in Live Mammalian Cells. Angew. Chem. Int. Ed. Engl. 59, 4511–4518 (2020).
Cawte, A.D., Unrau, P.J. & Rueda, D.S. Live cell imaging of single RNA molecules with fluorogenic Mango II arrays. Nat. Commun. 11, 1283 (2020).
Sunbul, M. et al. Super-resolution RNA imaging using a rhodamine-binding aptamer with fast exchange kinetics. Nat. Biotechnol. 39, 686–690 (2021).
Buhler, B. et al. Avidity-based bright and photostable light-up aptamers for single-molecule mRNA imaging. Nat. Chem. Biol. 19, 478–487 (2023).
Wu, J. et al. Live imaging of mRNA using RNA-stabilized fluorogenic proteins. Nat. Methods 16, 862–865 (2019).
O'Connell, M.R. et al. Programmable RNA recognition and cleavage by CRISPR/Cas9. Nature 516, 263–266 (2014).
Nelles, D.A. et al. Programmable RNA tracking in live cells with CRISPR/Cas9. Cell 165, 488–496 (2016).
Yang, L.Z. et al. Dynamic imaging of RNA in living cells by CRISPR-Cas13 systems. Mol. Cell 76, 981–997 (2019).
Wang, H. et al. CRISPR-mediated live imaging of genome editing and transcription. Science 365, 1301–1305 (2019).
Colognori, D., Trinidad, M. & Doudna, J.A. Precise transcript targeting by CRISPR-Csm complexes. Nat. Biotechnol. (2023).
Nishimasu, H. et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156, 935–949 (2014).
Jiang, F.G., Zhou, K.H., Ma, L.L., Gressel, S. & Doudna, J.A. A Cas9-guide RNA complex preorganized for target DNA recognition. Science 348, 1477–1481 (2015).
Smargon, A. A., et al. Crosstalk between CRISPR-Cas9 and the human transcriptome. Nat. Commun. 13, 1125 (2022).
Ma, H. et al. CRISPR-Cas9 nuclear dynamics and target recognition in living cells. J. Cell Biol. 214, 529–537 (2016).
Hendel, A. et al. Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat. Biotechnol. 33, 985–989 (2015).
Chen, B. et al. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 155, 1479–1491 (2013).
Jinek, M. et al. A Programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).
Konermann, S. et al. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 517, 583–588 (2015).
Li, N., Chang, C., Pan, W. & Tang, B. A multicolor nanoprobe for detection and imaging of tumor-related mRNAs in living cells. Angew. Chem. Int. Ed. Engl. 51, 7426–7430 (2012).
Hsu, P.D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832 (2013).
Ma, H. et al. CRISPR-Sirius: RNA scaffolds for signal amplification in genome imaging. Nat. Methods 15, 928–931 (2018).
Foekens, J.A., Romain, S., Look, M.P., Martin, P.M. & Klijn, J.G.M. Thymidine kinase and thymidylate synthase in advanced breast cancer: Response to tamoxifen and chemotherapy. Cancer Res. 61, 1421–1425 (2001).
Zhong, G. et al. A reversible RNA on-switch that controls gene expression of AAV-delivered therapeutics in vivo. Nat. Biotechnol. 38, 169–175 (2020).
Clemson, C.M. et al. An architectural role for a nuclear noncoding RNA: NEAT1 RNA is essential for the structure of paraspeckles. Mol. Cell 33, 717–726 (2009).
Wang, Y. et al. Genome-wide screening of NEAT1 regulators reveals cross-regulation between paraspeckles and mitochondria. Nat. Cell Biol. 20, 1145–1158 (2018).
Yamazaki, T. et al. Functional domains of NEAT1 architectural lncRNA induce paraspeckle assembly through phase separation. Mol. Cell 70, 1038–1053 (2018).
Chavez, A. et al. Highly efficient Cas9-mediated transcriptional programming. Nat. Methods 12, 326–328 (2015).

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 (25 mM), dNTP (10 mM), 6×DNA Loading Dye, mouse anti-β-actin (D191047), rabbit anti-MUC4 (D164414), rabbit anti-TK1 (D151614), 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). IRDye conjugated goat anti-mouse IgG (926-32210) and goat anti-rabbit IgG (926-32211) were purchased from LI-COR (Lincoln, Nebraska, USA). 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 1-5) 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 (100 nM) were annealed by heating to 95 ^oC, followed by cooling to room temperature. Pre-annealed agRNAs were hybridized with or without TK1 RNA (100 nM) 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 (100 nM) 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 (200 nM) or agRNAs (200 nM) with or without TK1 RNA (200 nM) were incubated with Cas9 protein (200 nM) 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, egRNA 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 was constructed with a mammalian expression lentiviral vector. cDNAs of miniCMV promoter, exogenous RNA, and 24×MS2 were obtained by overlap PCR and cloned into the MfeI and AgeI sites. By replacing miniCMV with miniP, a plasmid for expressing an exogenous RNA-24×MS2 tag with a weaker promoter was obtained. For specificity study, exogenous RNA plasmids without agRNA-targeting sequence or 24×MS2 tag were constructed. Plasmids for expressing MCP-GFP-NLS fusion protein and MCP-mCherry-NLS fusion protein under a CMV promoter was constructed with a mammalian expression vector pcDNA3.1(+). cDNAs of MCP-GFP-NLS or MCP-mCherry-NLS were cloned into HindIII and EcoRI sites. Plasmid for expressing an ER-targeting mRNA under a miniCMV promoter was constructed with a mammalian expression lentiviral vector. cDNAs of CytERM, GFP and the agRNA-targeting sequence were obtained by overlap PCR and cloned into NheI and BamHI 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, the cDNA of the corresponding responsive sequence (Supplementary Table 3) was obtained and cloned into the plasmid agRNA-24×SRB2 by seamless cloning. To image endogenous mRNAs, the target site is chosen at an accessible, single-stranded region at 3’ UTR or the coding sequence, whereas a single-stranded region far-away from the interacting motifs is selected for long non-coding RNA. To image MUC4 mRNA, β-actin mRNA and lncRNA NEAT1, the cDNAs of the corresponding responsive sequences for the target sites (Supplementary Table 3) were obtained and cloned into the plasmid agRNA-24×SRB2 by seamless cloning. To image c-myc and GalNAc-T, the cDNAs of the corresponding responsive sequences for the target sites (Supplementary Table 3) were obtained and cloned into agRNA-3×Mango and agRNA-3×Pepper, respectively.

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 expressing 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 backbone⁴⁵. For inducible transcription, DNA sequences with one, two or 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. For inducible transcription of endogenous genes, plasmids for expressing β-actin-responsive agRNAs with spacer sequences targeting ASCL1 and IL1RN, TK1-responsive agRNAs with spacer sequences targeting IL2 and IFN-γ, c-myc-responsive agRNAs with spacer sequences targeting IL2, and TK1- responsive agRNAs with spacer sequences targeting IFN-γ were obtained by cloning the cDNAs into the Addgene plasmid (#60955) at the BstxI and XhoI restriction sites under a U6 promoter.

Cell culture, plasmid transfection and live imaging. 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 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. The cells were transfected with a mixture of plasmids (0.2 μg/plasmid) as indicated and Lipofectamine 3000 (0.5 μL/plasmid) in Opti-MEM medium for 4 h according to the manufacturer’s instructions. Cells were cultured in complete medium and 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, egRNA 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 Act D (5 μg/mL) 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 test the ability of LUSTER system for multiplexed RNA imaging, different cells were transfected with dCas9, c-myc-, TK1- and GalNAc-T-responsive LUSTER system expressing agRNA-3×Mango, agRNA-3×SRB2 and agRNA-3×Pepper, respectively. Specificity was verified by depleting c-myc, TK1 or GalNAc-T in MCF-7 cells with corresponding siRNAs (Supplementary Table 3).

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. The specificity for the LUSTER system in imaging target RNAs with different localizations was verified by the corresponding FISH probes for the 5S-, 7SK- or CytERM- tagged RNA (Supplementary Table 4).

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 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 sodium arsenite (500 µM) 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 (500 µM, 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.

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 on a confocal laser scanning fluorescence microscope (Nikon, Eclipse TE2000-E) using a 60× oil objective lens (NA = 1.49) with a pinhole of 128 µm and an integration time of 2.5 µs. 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 imaging with LUSTER system. To test the ability of the LUSTER system to single molecule RNA imaging, MCF-7 cells were transfected with plasmids expressing dCas9 and agRNA-24×SRB2 responsive to the exogenous RNA or a non-repetitive region of MUC4 mRNA. The fluorescence images were obtained upon incubation with TMR-DN (50 nM) 24 h after transfection. The ability of the aptamer-dye based labeling system for single molecule imaging relies on the affinity (or dissociation constant K_d), and the working concentration of the dye, and the activation ratio (AR) of the aptamer-dye complex versus the dye, which can be discussed as follows: In the case of single molecule imaging, the dye is in much excess to target RNA, and the dye working concentration is usually set to be comparable to K_d (K_d » [dye]). Based on the definition of K_d,

K_d = [aptamer] [dye] / [aptamer-dye complex]

Then, we have [aptamer] » [aptamer-dye complex]

We can roughly estimate a labeling efficiency (LE) of ~50% for 24×SRB2, implying the formation of ~12 aptamer-dye complex in single-molecule RNA. To ensure reliably detection of single-molecule RNA with 24× tag, a signal-to-background ratio of ~10 is preferred. Assuming a typical detection volume of 0.5 fL in a confocal imaging system, the number of free dye molecules N_dye in the detection volume is

N_dye = 3 × 10⁸ ×[dye]

The signal-to-background ratio (SBR) for single-molecule RNA with 24×tag is given by

SBR = 12 × AR / N_dye = 4 × 10^-8 × AR / [dye] ³ 10

We obtain

K_d » [dye] £ 4 × 10^-9 × AR

This equation defines the optimal affinity for the aptamer-dye component of the labeling system in single-molecule RNA imaging. In the LUSTER system, SRB2 exhibited a K_d of ~36 nM toward TMR-DN with an AR of 17^13,20. The working concentration for TMR-DN was set to be 50 nM. First, the labeling efficiency was estimated to be ~50% for 24×SRB2 with ~12 aptamer−dye complex for single-molecule RNA. Second, assuming a typical detection volume of 0.5 fL in a confocal imaging system, there was ~15 free dye molecule (N_dye) in the detection volume. Third, the signal-to-background ratio for the single-molecule RNA is estimated by AR × 12 / N_dye = 13.6. Therefore, it is possible for us to achieve single-molecule RNA detection using 24×SRB2 tag.

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 the LUSTER system for tracking single-molecule translocation, MCF-7 cells were transfected with plasmids for the ER-targeting mRNA and the LUSTER system. To release the ER-targeting mRNA, cells were incubated with a translation inhibitor puromycin (100 μg/mL). Single-molecule translocation tracking was acquired for 30 s with an exposure time of 500 ms (0.5 s per frame). MTrack2 ImageJ plugin was used for single particle tracking in live cells.

Fluorescence images were obtained on a confocal laser scanning fluorescence microscope (Nikon, Eclipse TE2000-E) using a 60× oil objective lens (NA = 1.49) with a pinhole of 46 µm and an integration time of 2.5 µs. Fluorescence images were acquired using the following collection ranges upon excitation with lasers of different wavelengths. green channel: excitation: 488 nm, collection range: 500 - 550 nm, red channel: excitation: 561 nm, collection range: 575 - 625 nm. 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.

Inducible transcription activation by LUSTER system. To test the ability of RNA inducible transcription, MCF-7 or MCF-10A cells were cotransfected with plasmids expressing dCas9-VPR and agRNA-iRFP responsive to TK1 mRNA, fluorescence images were obtained at different times. To test the ability of LUSTER system to induce transcription of endogenous genes, HEK293T cells were cotransfected with plasmids expressing dCas9-VPR and β-actin-responsive agRNAs with spacer sequences targeting one or two promoter regions of ASCL1 and IL1RN. MCF-7 cells were cotransfected with plasmids expressing dCas9-VPR and TK1-responsive agRNAs with spacer sequences targeting one or two promoters of IL2 and IFN-γ. To test the ability of LUSTER system for orthogonal transcription activation, different cells were co-transfected with dCas9-VPR, c-myc- and TK1-responsive agRNAs with spacer sequences targeting one or two promoters of IL2 and IFN-γ, respectively. The specificity was verified by depleting c-myc or TK1 GalNAc-T in MCF-7 cells via transfection of 0.1 ng corresponding siRNAs using lipofectamine 2000 according to the manufacturer’s instructions. The expression levels of ASCL1, IL1RN, IL2 and IFN-γ were determined by qRT-PCR.

Single molecule RNA FISH. The LUSTER system in single-molecule RNA imaging was verified using the smFISH method according to the literature with slight modifications⁴⁶.Specifically, primary probes comprising a region for hybridization with Cy5-labeled secondary probes were designed for different target RNAs including 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 (50 nM) in PBS for 5 min. Fluorescence images were obtained with a Nikon TI-E+A1 SI confocal laser scanning microscope using a 60× oil immersion objective lens (NA = 1.49) with a pinhole of 46 µm and an integration time of 2.5 µs.

Quantitative RT-PCR (qRT-PCR) determination of RNA. Concentrations of RNAs including TK1, c-myc, gRNAs, HHR-cleaved agRNA-3×SRB2, β-actin, NEAT1, ASCL1, IL1RN, IL2 and IFN-γ under different conditions were determined by qRT-PCR using GAPDH as an internal reference according to previous studies⁴⁷. 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.

To quantify the expression of RNAs, a calibration curve for Ct values versus gene concentrations were obtained by qRT-PCR analysis of a set of standard samples of plasmids in serial dilution ratios. The copy numbers of RNAs under different conditions were determined according to the calibration curve.

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, rabbit anti-MUC4 or rabbit anti-TK1) 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 561 nm laser was used for SRB2 excitation and fluorescence emission was collected with a 610/20 nm band-pass filter. Cell doublets were removed using forward versus side scatter gating (Supplementary Fig. 21). SRB2 fluorescence gating was performed using the signals for untransfected cells as a negative control (Only subsets of cells with fluorescence higher untransfected cells were selected, and the histograms of the cell numbers versus fluorescence intensities were plotted). Flow cytometry data were analysed using FlowJo 7.6.

Data and statistical analysis. In single molecule RNA imaging assays, foci detection for GFP or mCherry from MS2–MCP method, SRB2 from the LUSTER system and Cy5 from smFISH probes were limited to foci at least 1.5-fold brighter than background signal. In β-actin imaging, foci detection of SGs were limited to foci at least 2-fold brighter than background signal. In NEAT1 imaging, foci detection of paraspeckles was limited to foci at least 2-fold brighter than background signal. Foci fluorescence intensity, sizes and pathways were analyzed using Fiji. For PCC calculation, the images were processed using the filters (1.5-fold of background signal for the single molecule RNA imaging, and 2-fold of background signal for β-actin and NEAT1 imaging) in the Fiji/ImageJ software. The foci were obtained using the Find Maxima function in the software. The scatter plots for the corresponding two fluorescence channels were created and the PCCs were calculated using the Scatter J plugin. Fiji software was used to analyze other microscopic images. Microsoft Excel, OriginPro and GraphPad Prism were used to analyze data and prepare figures. 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. 1c, 2d,g,h,j, 3d-f,h-j,n, 4c,d,g,h, and 5d,f-h and Extended Data Figs. 2c-e, 5a,c,d,f, 6b,e, 7c, 8b, 9b,d, 10a,b,d and Supplementary Figs. 5b-d, 6a, 7a-d, 9c,d, 12b, 14a,b,d,e, 16a,b, 17c,d, 18b-d, 19b 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

45. 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).

46. Tsanov, N. et al. smiFISH and FISH-quant – a flexible single RNA detection approach with super-resolution capability. Nucleic Acids Res.44, e165 (2016).

47. Nolan, T., Hands, R.E. & Bustin, S.A. Quantification of mRNA using real-time RT-PCR. Nat. Protoc.1, 1559‒1582 (2006).

There is NO Competing Interest.

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

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