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Endogenous promoter-driven sgRNA for monitoring the expression of low-abundance transcripts and lncRNAs

Abstract

Detection of endogenous signals and precise control of genetic circuits in the natural context are essential to understand biological processes. However, the tools to process endogenous information are limited. Here we developed a generalizable endogenous transcription-gated switch that releases single-guide RNAs in the presence of an endogenous promoter. When the endogenous transcription-gated switch is coupled with the highly sensitive CRISPR-activator-associated reporter we developed, we can reliably detect the activity of endogenous genes, including genes with very low expression (<0.001 relative to Gapdh; quantitative-PCR analysis). Notably, we could also monitor the transcriptional activity of typically long non-coding RNAs expressed at low levels in living cells using this approach. Together, our method provides a powerful platform to sense the activity of endogenous genetic elements underlying cellular functions.

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Fig. 1: Development of an endogenous switch by driving sgRNA expression directly from an endogenous promoter.
Fig. 2: SPH-OminiCMV-Ents can track the expression dynamics of low-abundance transcripts during differentiation.
Fig. 3: Detection of lncRNAs with SPH-OminiCMV-Ents.
Fig. 4: Encoding of multiple sgRNAs improves the sensitivity of SPH-OminiCMV-Ents and enables multiplexed transcriptional regulation.
Fig. 5: Homogeneous expression of mCherry by driving dCas9 and activators under a single promoter.
Fig. 6: Quantitative characterization of SPH (single CAG)-OminiCMV-Ents.

Data availability

The previously published RNA sequencing data that were re-analysed here are available under the accession code GSM2573084. All of the raw data associated with the figures are listed in the Source data (statistical source data). All of the raw images for the western blots can be found in the Source data (unprocessed western blots and gels). The sequences of all vectors are provided in Supplementary Fig. 1. All other materials and data are available on request. Source data are provided with this paper.

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Acknowledgements

We thank L. Quan, H. Wu and S. Qian from the FACS facility in ION as well as Y. Wang, Y. Zhang, X. Chen, D. Xiang and Q. Hu from the Optical Imaging facility. We thank N. Zhong and Q. Wang for their technical assistance. This work was supported by the Basic Frontier Scientific Research Program of the Chinese Academy of Sciences From 0 to 1 original innovation project (grant no. ZDBS-LY-SM001), R&D Program of China (grant nos 2017YFC1001300 and 2018YFC2000100), CAS Strategic Priority Research Program (grant no. XDB32060000), National Natural Science Foundation of China (grant nos 31871502, 31925016, 91957122 and 31901047), Shanghai Municipal Science and Technology Major Project (grant no. 2018SHZDZX05), Shanghai City Committee of Science and Technology Project (grant nos 18411953700, 18JC1410100 and 19XD1424400) and International Partnership Program of Chinese Academy of Sciences (grant no. 153D31KYSB20170059).

Author information

Affiliations

Authors

Contributions

N.G. designed experiments, constructed vectors, performed transfections, generated cell lines and analysed data. J.Hu performed RNA FISH, immunofluorescence staining and assisted with the vector construction. B.H., J.Huang, Yu Wei, J.P., Yinghui Wei, X.S. and L.S. assisted with the generation of cell lines and analysis. Z.J. constructed vectors and performed qPCR analysis and cell-line genotyping. X.H., Q.X. and H.L. performed the transient transfections and analysis in the cell cultures. N.G. and X.F. performed the western blots. Y.S. and Y.Z. designed synthetic sgRNA sequences and analysed the public RNA sequencing data. C.Z. assisted with the vector construction. H.Z. and H.Y. conceived the project, designed experiments, supervised the project and wrote the paper.

Corresponding authors

Correspondence to Haibo Zhou or Hui Yang.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Cell Biology thanks Rory Johnson, Ophir Shalem and the other, anonymous, reviewers for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Mean mCherry intensity induced by different sgRNAs and Optimization of the miniCMV promoter.

a, The fluorescence intensity of mCherry induced by different sgRNAs (n = 2 repeats). Note that the intensity was quantified by FACS, 24 hours after transient transfection of SPH, miniCMV and sgRNA in 293T cells. b, Representative images showing mCherry expression in N2a cells induced by different mini-promoters, 24 hours after transient transfection, each experiment was independently repeated 3 times with similar results. Scale bar, 200 μm. c, Mean fluorescence intensity of mCherry, 48 hours after transient transfection, n = 3 repeats per group (miniCMV v.s. Mini-TK, p < 0.0001; miniCMV v.s. Luc2CP, p < 0.0001; miniCMV v.s. TRE3G, p < 0.0001; unpaired two-sided Student’s t test). d, Mean mCherry intensity induced by different TS intervals, 48 hours after transient transfection, n = 3 repeats per group. e, The influence of sgRNA copy number on mCherry expression for different TS intervals, number above the bar indicates the number of repeats per group. f, Mean fluorescence intensity of mCherry induced by SPH-OminiCMV and different promoters, 48 hours after transient transfection. n = 3 repeats per group. g, Representative images showing that SPH-OminiCMV induces higher levels of mCherry than commonly used strong promoters. The tagBFP was co-transfected to control the transfection efficiency. Experiments were independently repeated 2 times per group with similar results. Scale bar: 50 μm. N2a cells were transiently transfected with plasmids for all experiments. All values are presented as mean ± s.e.m.; unpaired two-sided Student’s t test; *p < 0.05, **p < 0.01, ***p < 0.001. Statistical source data are provided in Source Data Extended Data Fig. 1. Source data

Extended Data Fig. 2 SPH-OminiCMV induced higher levels of gene expression than SPH-mediated endogenous activation and CMV-mediated overexpression.

a, Schematic showing SPH-mediated endogenous gene activation, CMV- and SPH-OminiCMV-mediated exogenous gene expression. b, Relative mRNA expression levels of different genes, n = 2 repeats per group. Statistical source data are provided in Source Data Extended Data Fig. 2. Source data

Extended Data Fig. 3 Specificity of SPH-OminiCMV and generation of SPH-OminiCMV transgenic mESCs.

a, Tolerance of improved reporter system to mismatched sgRNAs by transient transfection of SPH, OminiCMV and sgRNA in N2a cells. X-axis: No. of sgRNA TS; Y-axis: No. of sgRNA. Scale bar indicates the mean fluorescence intensity (red, high; white, low). The sgRNA information is provided in Supplementary Table 1, n = 2 repeats per group. b, Schematic illustration of the transgenes. c, Genotyping of SPH-OminiCMV transgenic mESCs via PCR, ‘+’ indicates the positive colony and ‘-’ indicates the negative colony, images are representative of several ‘+’ colonies. d, Images showing transient expression of sgRNA induced mCherry expression in the SPH-OminiCMV positive colony, images are representative of 3 experiments. Scale bar: 200 μm. e, Relative mCherry expression quantified by qPCR, n = 3 repeats per group. All values are presented as mean ± s.e.m.. Statistical source data and unprocessed gels are provided in Source Data Extended Data Fig. 3. Source data

Extended Data Fig. 4 Targeted insertion of tRNA-sgRNA-tRNA into the 3’UTR does not affect the normal protein production of target genes.

a, Schematic showing that tRNA-sgRNA-tRNA was inserted into the 3’UTR or intron of Actb, and mean fluorescence intensity of mCherry. Number above the bar indicates the number of repeats per group (SPH-OminiCMV-Ents-Intron 1 v.s. SPH-OminiCMV-Ents-3’UTR, p = 0.5689; unpaired two-sided Student’s t test). b, Western blots, n = 4 repeats per group. c, Quantification of Western blots data showing that insertion of tRNA-sgRNA-tRNA into the 3’UTR of Actb loci did not influence the production of Actb (SPH-OminiCMV-Ents v.s. SPH-OminiCMV, p = 0.3456; SPH-OminiCMV-Ents v.s. P2A-mCherry, p = 0.6301; unpaired two-sided Student’s t test). Number above the bar indicates the number of repeats per group. d, e, Insertion of the tRNA-sgRNA-tRNA targeting LacZ in SPH-OminiCMV mESCs does not induce mCherry expression, number above the bar indicates the number of repeats per group. All values are presented as mean ± s.e.m.; unpaired two-sided Student’s t test; *p < 0.05, **p < 0.01, ***p < 0.001. Statistical source data and unprocessed western blots are provided in Source Data Extended Data Fig. 4. Source data

Extended Data Fig. 5 SPH-OminiCMV-Ents enables the visualization of low-abundance genes during cell differentiation, and insertion of an sgRNA array into the non-expressed gene does not induce mCherry expression.

a, b, Representative images showing mCherry expression during differentiation and quantification of Esrrb mRNA levels using qPCR in SPH-OminiCMV-Ents-Esrrb mESCs during differentiation. Scale bar, 50 μm; n = 4 repeats per group. c, d, Representative images showing mCherry expression and quantification of Sox2 mRNA levels using qPCR in SPH-OminiCMV-Ents-Sox2 mESCs during differentiation. Scale bar, 50 μm; n = 4 repeats per group. e, f, Representative images showing mCherry expression and quantification of Tet1 mRNA levels using qPCR in SPH-OminiCMV-Ents-Tet1 mESCs during differentiation. Scale bar: 50 μm, n = 4 repeats per group. g, Schematic showing insertion of one sgRNA or an sgRNA array into the 3’UTR of Sema3a locus. h, Quantification of Sema3a expression by qPCR in ESCs, n = 3 repeats. i, Images showing mCherry expression, images are representative of 4 experiments. Scale bar, 50 μm. j, Mean mCherry fluorescence intensity. Number above the bar indicates the number of colonies per group (SPH-OminiCMV-Ents-one sgRNA v.s. SPH-OminiCMV, p = 0.5964; SPH-OminiCMV-Ents-sgRNA array v.s. SPH-OminiCMV, p = 0.0761; unpaired two-sided Student’s t test). All values are presented as mean ± s.e.m.; unpaired two-sided Student’s t test; *p < 0.05, **p < 0.01, ***p < 0.001. Statistical source data are provided in Source Data Extended Data Fig. 5. Source data

Extended Data Fig. 6 Homogeneous expression of mCherry by driving dCas9 and activators under a single promoter.

a, Schematic of the vector. Note that dCas9 and P65-HSF1 were expressed by two CAG promoters respectively. b, Histogram of FACS analysis of a SPH-OminiCMV-Ents-Actb colony. c, Different expression levels of activators between mCherry-high and mCherry-low cells from the same SPH-OminiCMV-Ents-Actb colony, n = 3 repeats (High-5% v.s. Low-5%: dCas9, p = 0.0028; p65-HSF1, p = 0.9161; Actb, p = 0.0743; sgRNA, p = 0.0030; unpaired two-sided Student’s t test). d, Schematic showing SPH (single CAG), note that the expression of dCas9 and p65-HSF1 was driven by a single promoter. e, Histogram of FACS analysis of a SPH (single CAG) -OminiCMV-Ents-Actb colony. All values are presented as mean ± s.e.m.; unpaired two-sided Student’s t test; *p < 0.05, **p < 0.01, ***p < 0.001. Statistical source data are provided in Source Data Extended Data Fig. 6. Source data

Extended Data Fig. 7 FACS analysis of mCherry expression.

a, Analysis of the flow cytometry data showing how R1 was gated (side scatter: SSC; forward scatter: FSC). b, Representative histogram of FACS analysis of SPH (single CAG)-OminiCMV-Ents-one sgRNA, SPH (single CAG)-OminiCMV-Ents-sgRNA array and P2A-mCherry cells for different genes. Each experiment was independently repeated several times with similar results. For the number of repeats, see Fig. 5d. c, Representative histogram of SPH (single CAG)-OminiCMV-Ents-one sgRNA and SPH (single CAG)-OminiCMV-Ents-sgRNA array cells for different lncRNAs. Each experiment was independently repeated several times with similar results. For the number of repeats, see Fig. 5f.

Extended Data Fig. 8 The side-by-side comparison of different strategies and downregulation of mCherry and Nanog at the protein level during differentiation.

a, Analysis of the flow cytometry data showing how R1 was gated (side scatter: SSC; forward scatter: FSC). b, Representative histogram of FACS analysis of SPH (single CAG)-OminiCMV-Ents-one sgRNA, SPH (single CAG)-OminiCMV-Ents-sgRNA array and P2A-mCherry cells for different genes. Each experiment was independently repeated several times with similar results. For the number of repeats, see Fig. 5d. c, Representative histogram of SPH (single CAG)-OminiCMV-Ents-one sgRNA and SPH (single CAG)-OminiCMV-Ents-sgRNA array cells for different lncRNAs. Each experiment was independently repeated several times with similar results. For the number of repeats, see Fig. 5f.

Extended Data Fig. 9 SPH (single CAG)-OminiCMV-Ents-sgRNA array induces the highest expression of mCherry.

a, Comparison of the fluorescence intensity of SPH-OminiCMV-Ents-one sgRNA, SPH-OminiCMV-Ents-sgRNA array, SPH (single CAG)-OminiCMV-Ents-one sgRNA and SPH (single CAG)-OminiCMV-Ents-sgRNA array systems. Number in the bar indicates the number of colonies per group. Unpaired two-sided Student’s t test. Note that SPH (single CAG)-OminiCMV-Ents-one sgRNA and SPH (single CAG)-OminiCMV-Ents-sgRNA array data are also shown in Fig. 5d; SPH-OminiCMV-Ents-one sgRNA and SPH-OminiCMV-Ents-sgRNA array data are also shown in Figs. 2c, 4e. b, LncRNA expression matrix of embryonic stem cells. Note that the data was downloaded from the public GEO database (GSM2573084) and lncRNAs were ordered according to their expression levels in a decreasing order. LncRNAs marked in red and black indicate those that explored in Fig. 5f. In total, 14432 lncRNAs were detected with a FPKM value higher than 0 and 9640 lncRNAs have a FPKM value higher than that of Pvt1. All values are presented as mean ± s.e.m.; unpaired two-sided Student’s t test; *p < 0.05, **p < 0.01, ***p < 0.001. Statistical source data and p values for a are provided in Source Data Extended Data Fig. 9. Source data

Supplementary information

Supplementary Information

Supplementary sequences.

Reporting Summary

Supplementary Tables

Supplementary Table 1: sgRNA sequences. Supplementary Table 2: sgRNA sequences for endogenous gene activation. Supplementary Table 3: The sgRNA sequences for inserting the sgRNA precursor. Supplementary Table 4: Primers for identifying the sgRNA insertion. Supplementary Table 5: Relative expression levels. Supplementary Table 6: qPCR primers.

Source data

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Gao, N., Hu, J., He, B. et al. Endogenous promoter-driven sgRNA for monitoring the expression of low-abundance transcripts and lncRNAs. Nat Cell Biol 23, 99–108 (2021). https://doi.org/10.1038/s41556-020-00610-9

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