Abstract
RNA is a central and universal mediator of genetic information underlying the diversity of cell types and cell states, which together shape tissue organization and organismal function across species and lifespans. Despite numerous advances in RNA sequencing technologies and the massive accumulation of transcriptome datasets across the life sciences1,2, the dearth of technologies that use RNAs to observe and manipulate cell types remains a bottleneck in biology and medicine. Here we describe CellREADR (Cell access through RNA sensing by Endogenous ADAR), a programmable RNA-sensing technology that leverages RNA editing mediated by ADAR to couple the detection of cell-defining RNAs with the translation of effector proteins. Viral delivery of CellREADR conferred specific cell-type access in mouse and rat brains and in ex vivo human brain tissues. Furthermore, CellREADR enabled the recording and control of specific types of neurons in behaving mice. CellREADR thus highlights the potential for RNA-based monitoring and editing of animal cells in ways that are specific, versatile, simple and generalizable across organ systems and species, with wide applications in biology, biotechnology and programmable RNA medicine.
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Data availability
The RNA-seq data are available at NCBI BioProject under accession code PRJNA856413. Genome reference data are available from Gencode GRCH38.p13. Plasmids will be deposited to Addgene. We will provide reagents upon request until they are available from Addgene.
Code availability
The codes for optogenetic activation analyses are available from https://github.com/XuAn-universe/Optogenetic-activation. The codes for fibre photometry analyses are available from https://github.com/XuAn-universe/Fiber-photometry-sensory-stimulation.
References
Hu, B. C. The human body at cellular resolution: the NIH Human Biomolecular Atlas Program. Nature 574, 187–192 (2019).
BRAIN Initiative Cell Census Network (BICCN). A multimodal cell census and atlas of the mammalian primary motor cortex. Nature 598, 86–102 (2021).
Arendt, D. et al. The origin and evolution of cell types. Nat. Rev. Genet. 17, 744–757 (2016).
Regev, A. et al. The Human Cell Atlas. eLife 6, e27041 (2017).
Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).
Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).
Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).
Baylis, F., Darnovsky, M., Hasson, K. & Krahn, T. M. Human germ line and heritable genome editing: the global policy landscape. CRISPR J. 3, 365–377 (2020).
Feng, G. et al. Opportunities and limitations of genetically modified nonhuman primate models for neuroscience research. Proc. Natl Acad. Sci. USA 117, 24022–24031 (2020).
Mich, J. K. et al. Functional enhancer elements drive subclass-selective expression from mouse to primate neocortex. Cell Rep. 34, 108754 (2021).
Hartl, D., Krebs, A. R., Juttner, J., Roska, B. & Schubeler, D. Cis-regulatory landscapes of four cell types of the retina. Nucleic Acids Res. 45, 11607–11621 (2017).
Juttner, J. et al. Targeting neuronal and glial cell types with synthetic promoter AAVs in mice, non-human primates and humans. Nat. Neurosci. 22, 1345–1356 (2019).
Nair, R. R., Blankvoort, S., Lagartos, M. J. & Kentros, C. Enhancer-driven gene expression (EDGE) enables the generation of viral vectors specific to neuronal subtypes. iScience 23, 100888 (2020).
Vormstein-Schneider, D. et al. Viral manipulation of functionally distinct interneurons in mice, non-human primates and humans. Nat. Neurosci. 23, 1629–1636 (2020).
Graybuck, L. T. et al. Enhancer viruses for combinatorial cell-subclass-specific labeling. Neuron 109, 1449–1464.e13 (2021).
Dimidschstein, J. et al. A viral strategy for targeting and manipulating interneurons across vertebrate species. Nat. Neurosci. 19, 1743–1749 (2016).
Eisenberg, E. & Levanon, E. Y. A-to-I RNA editing—immune protector and transcriptome diversifier. Nat. Rev. Genet. 19, 473–490 (2018).
Nishikura, K. A-to-I editing of coding and non-coding RNAs by ADARs. Nat. Rev. Mol. Cell Biol. 17, 83–96 (2016).
Abudayyeh, O. O. et al. A cytosine deaminase for programmable single-base RNA editing. Science 365, 382–386 (2019).
Katrekar, D. et al. In vivo RNA editing of point mutations via RNA-guided adenosine deaminases. Nat. Methods 16, 239–242 (2019).
Merkle, T. et al. Precise RNA editing by recruiting endogenous ADARs with antisense oligonucleotides. Nat. Biotechnol. 37, 133–138 (2019).
Qu, L. et al. Programmable RNA editing by recruiting endogenous ADAR using engineered RNAs. Nat. Biotechnol. 37, 1059–1069 (2019).
Rauch, S. et al. Programmable RNA-guided RNA effector proteins built from human parts. Cell 178, 122–134.e12 (2019).
Tan, M. H. et al. Dynamic landscape and regulation of RNA editing in mammals. Nature 550, 249–254 (2017).
Beier, H. & Grimm, M. Misreading of termination codons in eukaryotes by natural nonsense suppressor tRNAs. Nucleic Acids Res. 29, 4767–4782 (2001).
Segel, M. et al. Mammalian retrovirus-like protein PEG10 packages its own mRNA and can be pseudotyped for mRNA delivery. Science 373, 882–889 (2021).
Yang, C. F. et al. Sexually dimorphic neurons in the ventromedial hypothalamus govern mating in both sexes and aggression in males. Cell 153, 896–909 (2013).
Ran, F. A. et al. Genome engineering using the CRISPR–Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).
Chung, H. et al. Human ADAR1 prevents endogenous RNA from triggering translational shutdown. Cell 172, 811–824.e14 (2018).
Lamers, M. M., van den Hoogen, B. G. & Haagmans, B. L. ADAR1: "editor-in-chief" of cytoplasmic innate immunity. Front. Immunol. 10, 1763 (2019).
Pertea, M., Kim, D., Pertea, G. M., Leek, J. T. & Salzberg, S. L. Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown. Nat. Protoc. 11, 1650–1667 (2016).
Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).
Lo Giudice, C., Tangaro, M. A., Pesole, G. & Picardi, E. Investigating RNA editing in deep transcriptome datasets with REDItools and REDIportal. Nat. Protoc. 15, 1098–1131 (2020).
Lodato, S. et al. Gene co-regulation by Fezf2 selects neurotransmitter identity and connectivity of corticospinal neurons. Nat. Neurosci. 17, 1046–1054 (2014).
Matho, K. S. et al. Genetic dissection of the glutamatergic neuron system in cerebral cortex. Nature 598, 182–187 (2021).
Hodge, R. D. et al. Conserved cell types with divergent features in human versus mouse cortex. Nature 573, 61–68 (2019).
Eugene, E. et al. An organotypic brain slice preparation from adult patients with temporal lobe epilepsy. J. Neurosci. Methods 235, 234–244 (2014).
Anzalone, A. V., Koblan, L. W. & Liu, D. R. Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors. Nat. Biotechnol. 38, 824–844 (2020).
Mitric, M. et al. Layer- and subregion-specific electrophysiological and morphological changes of the medial prefrontal cortex in a mouse model of neuropathic pain. Sci Rep. 9, 9479 (2019).
Huang, Z. J. & Paul, A. The diversity of GABAergic neurons and neural communication elements. Nat. Rev. Neurosci. 20, 563–572 (2019).
Yi, Z. et al. Engineered circular ADAR-recruiting RNAs increase the efficiency and fidelity of RNA editing in vitro and in vivo. Nat. Biotechnol. 40, 946–955 (2022).
Katrekar, D. et al. Efficient in vitro and in vivo RNA editing via recruitment of endogenous ADARs using circular guide RNAs. Nat. Biotechnol. 40, 938–945 (2022).
Monian, P. et al. Endogenous ADAR-mediated RNA editing in non-human primates using stereopure chemically modified oligonucleotides. Nat. Biotechnol. 40, 1093–1102 (2022).
Hou, X., Zaks, T., Langer, R. & Dong, Y. Lipid nanoparticles for mRNA delivery. Nat. Rev. Mater. 6, 1078–1094 (2021).
Bedbrook, C. N., Deverman, B. E. & Gradinaru, V. Viral strategies for targeting the central and peripheral nervous systems. Annu. Rev. Neurosci. 41, 323–348 (2018).
Joyce, J. A. & Fearon, D. T. T cell exclusion, immune privilege, and the tumor microenvironment. Science 348, 74–80 (2015).
Aquino-Jarquin, G. Novel engineered programmable systems for ADAR-mediated RNA editing. Mol. Ther. Nucleic Acids 19, 1065–1072 (2020).
Cox, D. B., Platt, R. J. & Zhang, F. Therapeutic genome editing: prospects and challenges. Nat. Med. 21, 121–131 (2015).
Doudna, J. A. The promise and challenge of therapeutic genome editing. Nature 578, 229–236 (2020).
Kaiser, T., Zhou, Y. & Feng, G. Animal models for neuropsychiatric disorders: prospects for circuit intervention. Curr. Opin. Neurobiol. 45, 59–65 (2017).
He, M. et al. Strategies and tools for combinatorial targeting of GABAergic neurons in mouse cerebral cortex. Neuron 92, 555 (2016).
Schwarz, N. et al. Human cerebrospinal fluid promotes long-term neuronal viability and network function in human neocortical organotypic brain slice cultures. Sci. Rep. 7, 12249 (2017).
Schwarz, N. et al. Long-term adult human brain slice cultures as a model system to study human CNS circuitry and disease. eLife 8, e48417 (2019).
Acknowledgements
We thank L. Wan for providing the HeLa cell line; M. Tadross, A. West, K. Meyer, A. Zador and S. Soderling for comments on the manuscript; J. Hatfield and B.-x. Han for animal preparation; and R. Utama for bioinformatic analysis. This work was supported in part by NIMH grants 1DP1MH129954-01 and 5U19MH114821-03 to Z.J.H. D.G.S. was supported by the NINDS K12 Neurosurgery Research Career Development Program K12 Award and the Klingenstein-Simons Foundation. Extended Data Fig. 12a was generated using BioRender.
Author information
Authors and Affiliations
Contributions
Y.Q. and Z.J.H. conceived this study. Z.J.H. designed and supervised the research, analysed data and wrote the manuscript. Y.Q. designed the research, performed experiments, analysed data and wrote the manuscript. J.L. performed the FACS analysis, RNA-seq, quantitative PCR and western blotting. S.Z. generated AAV vectors. W.Z. and B.-S.W. validated CellREADR AAV vectors. X.A. performed neuron-type recording and manipulation experiments in behaving mice. X.Y. generated AAV vectors for rats. E.A.M., M.A., M.Y. and C.P. performed all human CellREADR experiments and analysed the data. D.G.S. designed and supervised the research involving human tissues, and provided input to the manuscript.
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Competing interests
Z.J.H. and Y.Q. have filed a provisional patent application on CellREADR technology through Duke University. The other authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Design and test of singular and binary CellREADR vectors.
a, Schematic of a singular CellREADR vector. Left, PGK-tdT (top) expresses the tdTomato target RNA from a PGK promoter. READRtdT-GFP (bottom) expresses a READR RNA consisting of sesRNAtdT and efRNAGFP, driven by a CAG promoter. Vertical dashed lines indicate the complementary base-pairing region between tdT mRNA and sesRNAtdT, with sequence surrounding the editable STOP codon shown on the right. At the editing site, the editable adenine in sesRNAtdT (cyan) is mismatched to a cytosine in the tdT mRNA. TdTomato is a tandem repeat of two dTomato genes, thus a tdT RNA contains two copies of target sequence for sesRNAtdT base-pairing. b, Validation of the READRtdT-GFP vector. In 293T cells co-transfected with READRtdT-GFP and PGK-tdT, many cells switched on GFP translation and fluorescence (arrows in top row). In cells co-transfected with control empty vector, few cells showed GFP expression (bottom right). c, GFP expression was further assayed by Western blotting. d, Left, a binary vector design for CellREADR luciferase assay. READRtdT-tTA2 expresses a readrRNA consisting of sesRNAtdT and efRNAtTA2, and TRE3g-ffLuc expresses the luciferase RNA upon tTA2 activation. Right, luciferase activity dramatically increased only in cells transfected with three vectors. Co-transfection of TRE3g-ffLuc with CAG-tTA2, which constitutively expresses tTA2, served as a positive control. e, Schematic of READRtdT-GFP vector in which a spacer sequence is inserted before sesRNAtdT coding region (top). 293T cells were transfected with READRtdT-GFP vector encoding viable length of spacers without (gray) or with (pink) tdT target RNA expression, respectively. Quantification of conversion ratio calculated as percentage of GFP+ cells among RFP+ cells (bottom). f, A binary vector design for CellREADR assay (left). Right, representative images of GFP conversion with binary vectors. In cells co-transfected with sesRNACtrl vector, few GFP+ cells were observed. Conversion percentages are shown on the right. Error bars in d are mean values ± s.e.m. n = 3 independent experiments performed. The bars in e are mean values, n = 2 independent experiments performed. For gel source data in c, see Supplementary Fig. 2.
Extended Data Fig. 2 CellREADR enables RNA sensing dependent gene editing and cell ablation, and Cre or Flp as an effort shows leaky activities.
a, left, vector design for CellREADR-mediated and target RNA-dependent gene editing. In READR tdT-Cas9/GFP, a CAG promoter drives expression of BFP followed by sequences coding for sesRNAtdT, Cas9, and eGFP effectors. In another vector, EF1a promoter drives tdT expression and U6b promoter drives the expression of a guide RNA (gRNA) targeting the DYRK1A gene in 293T cells. Right, quantification of READRtdT-Cas9/GFP efficiency as percent of GFP among RFP and BFP expressing cells with or without tdT target RNA. b, Cells transfected with the both U6b-gRNADYRK1A-CAG-tdT and READRtdT-Cas9/GFP showed robust GFP expression co-localized with BFP and RFP (bottom). Cells transfected with READR tdT-Cas9/GFP only (top) showed almost no GFP expression. c, SURVEYOR assay showed Cas9-mediated cleavage in the human DYRK1A locus. DNA cleavage was observed in cell lysates transfected with U6b-gRNADYRK1A-CAG-tdT and READR tdT-Cas9/GFP, but not in U6b-gRNADYRK1A and READRtdT-Cas9/GFP that lacked tdT target RNA. CAG-Cas9 with U6b-gRNADYRK1A cell lysate and 293T cell lysate without plasmid transfection were used as positive control and negative control, respectively. Arrows indicate cleavage products. d, left, vector design for CellREADR-mediated and target RNA-dependent cell death induction. In READR tdT-taCasp3-TEVp, a CAG promoter drives expression of BFP followed by sequences coding for sesRNAtdT and taCasp3-TEVp as effector to induce cell death. Right, cell apoptosis level measured by luminescence was increased in the cells transfected READR tdT-taCasp3-TEVp and EF1a-tdT compared with cells with no tdT RNA. e, Schematic READR vector with Cre coding sequence as effector RNA (left). Representative images of GFP conversion in CellREADR (right). In READRtdT-Cre vector without tdTom RNA, numerous cells showed GFP expression resulting from leaky CRE translation and recombination (right top). Cells transfected with READRtdT-Cre and tdTom RNA showed robust and strong GFP expression (right bottom). Cbh-DIO-eGFP was used as reporter vector for Cre. f, Schematic READR vector with Flp coding sequence as effector RNA (left). Representative images of GFP conversion in CellREADR (right). In READRtdT-Flp vector without tdTom RNA, numerous cells showed GFP expression from leaky FLP translation and recombination (right top). Cells transfected with READRtdT-Flp and tdTom RNA showed robust and strong GFP expression (right bottom). Cbh-fDIO-eGFP was used as reporter vector for Flp. Error bars in a and d are mean values ± s.e.m. n = 3 independent experiments performed.
Extended Data Fig. 3 Characterization of sesRNA properties.
a, CellREADR functions in multiple human and mouse cell lines. Schematic of CellREADR vectors. CAG-tdT expresses the tdTomato target RNA from a CAG promoter. READRtdT-GFP expresses a READR RNA consisting of sesRNAtdT and efRNAGFP, driven by a CAG promoter (top). Human (Hela) and mouse (N2a, KPC1242) cell lines showed comparable CellREADR efficiency. Error bars are mean values ± s.e.m. n = 3, n represents the number of independent experiments performed. b, Effects of different nucleotide mismatches between sesRNA and target RNA. Schematic of CellREADR vectors (top, related to Fig. 2b). CellREADR efficiency measured as RFP to GFP conversion ratio with different types of mismatch number, indel or two mismatches within UAG STOP codon. Indel here is one nucleotide insertion or deletion. Error bars are mean values ± s.e.m. n = 3, n represents the number of independent experiments performed. c, CellREADR mediated sensing of endogenous EEF1A1 mRNA and effector translation in 293T cells increased with longer incubation time after transfection. Error bars are mean values ± s.e.m. n = 3, n represents the number of independent experiments performed. d, Vector design of a tri-color CellREADR assay system (top). Quantification of READRtdT-GFP efficiency with increasing amount of CAG-tdT vector used for co-transfection of 293T cells (bottom). e, Schematics of rtTA-TRE3g-ChETA and READRChETA-GFP vectors (also see Fig. 1e). f, Representative images of co-transfected 293Tcells treated with different concentration of tetracycline in culture medium (also see Fig. 1e, f, g). g–h, Co-transfection of READRChETA-GFP with a vector that constitutively expresses ChETA (g) resulted highest conversion to GFP expression cells (h) compared to those in (f).
Extended Data Fig. 4 ADAR1 is necessary for CellREADR in 293T cells.
a, Schematic for generating a ADAR1 knockout cell line with CRISPR/Cas9. b, Western blot analysis showing ADAR1 expression in wild-type and no expression in ADAR1 knockout cells. c, Schematics of EF1a-ChETA-tdT and READRtdT-GFP vectors (left), and ADAR1 isoform expression vectors (right). d, Both p110 and p150 ADAR1 isoforms rescued the CellREADR functionality assayed by cell conversion ratio in ADAR1 knockout cells. e–h, INF-b increased CellREADR efficiency and ADAR expression. e, Schematic of EF1a-ChETA-tdT and READRtdT-GFP vectors. f, Western blot analysis showed increased expression of ADAR1-p110 protein and induction of ADAR1-p150 isoform after interferon treatment. g, Representative FACS analysis of GFP and RFP expression with mock (left) or interferon treatment (right). h, Quantification of the READRtdT-GFP efficiency in g, which was increased by interferon treatment. Error bars in d and h are mean values ± s.e.m. n = 3, n represents the number of independent experiments performed. For gel source data, see Supplementary Fig. 2.
Extended Data Fig. 5 Effects of CellREADR on targeted mRNA.
a, Quantitative PCR showing that CellREADR-mediated sesRNA expression did not impact the expression levels of targeted RNAs. Error bars are mean values ± s.e.m. n = 3 biological replicates performed. Ordinary one-way ANOVA followed by Dunnett's multiple comparisons with the mean of control was used for analysis. All the P values are indicated. b, Base-pairing of EEF1A1 mRNA and sesRNAEEF1A1-CDS. EEF1A1 mRNA or sesRNA was represented in blue and red, respectively. Peptide translated from EEF1A1 mRNA was highlighted in brown. Targeted region of EEF1A1 mRNA was analyzed by RNAseq. c, The ratios of A-to-G changes in EEF1A1 mRNA at each adenosine position was quantified and shown in heatmap in both sesRNAEEF1A1-CDS and sesRNACtrl samples. Two adenosines (A107 and A115) showed higher rate of A-to-G editing (c). Off-target editing of two sensitive adenosines can induce potential amino acid change (underlined in b). d, Base-pairing of PCNA mRNA and sesRNAPCNA. PCNA mRNA or sesRNA was represented in blue and red, respectively. Peptide translated from PCNA mRNA was highlighted in brown. Targeted region of PCNA mRNA was analyzed by RNAseq. e, The ratios of A-to-G changes in PCNA mRNA at each adenosine position was quantified and shown in heatmap in both sesRNAPCNA and sesRNACtrl samples. Three adenosines (A21, A29 and A129) showed higher rate of A-to-G editing. Off-target editing of three sensitive adenosines can induce potential amino acid change (underlined in d).
Extended Data Fig. 6 Design and screen of sesRNAs targeting Fezf2 and Ctip2 RNAs in vitro and in vivo.
a–b, Genomic structures of mouse Fezf2 (a) and Ctip2 (b) genes with locations of various sesRNAs as indicated. c, List of sesRNAs and Fezf2 and Ctip2 target gene fragments used for sesRNA screen.d, In Target vectors CAG-BFP-Fezf2 or CAG-BFP-Ctip2, a 200–3000 bp genomic region of the Fezf2 or Ctip2 gene containing sequences complementary to a sesRNA in a, b were cloned downstream to the BFP and T2a coding region driven by a CAG promoter. In READR vectors, READRFezf2-GFP or READRCtip2-GFP expresses corresponding sesRNAs shown in a, b. e–f, Quantification of efficiencies READRFezf2-GFP (e) or READRCtip2-GFP (f) as GFP conversion ratio by FACS assay of 293T cells co-transfected with CAG-BFP-Fezf2 or CAG-BFP-Ctip2 target vector, respectively. g, Schematic of binary READR AAV vectors. In READR vector, a hSyn promoter drives expression of mCherry followed by sequences coding for sesRNACtip2, smFlag and tTA2 effectors. In Reporter vector, TRE3g promoter drives mNeon in response to tTA2 from the READR vector. h, Coronal sections of mouse cortex injected with binary READRFezf2 vectors. mNeon indicated READRFezf2 labeled cells. Four Fezf2 sesRNAs were screened. i, Quantification of specificity of 4 Fezf2 sesRNAs in h. For Fezf2 sesRNA in-vivo screen, the specificity of each sesRNA was calculated by co-labeling by READR AAVs and CTIP2 antibody (due to lack of FEZF2 antibody); as Ctip2 represents a subset of Fezf2+ cells (not shown), CTIP2 antibody gives an underestimate of the specificity of Fezf2 sesRNA. SesRNA1 showed highest specificity. j, Coronal sections of mouse cortex injected with binary READRCtip2 vectors. mNeon indicated binary READR labeled cells. Eight Ctip2 sesRNAs were screened. k, Quantification of specificity of 8 sesRNAs in j. The specificity of each sesRNA was calculated by co-labeling by binary READRCtip2 AAVs and CTIP2 antibody (not shown). SesRNA3 and sesRNA8 showed highest specificity. Error bars in e–f are mean values ± s.e.m. n = 3, n represents the number of independent experiments performed. Bars in i and k are values from one mouse performed. Each bar in i and k is the value from one mouse performed (n = 1).
Extended Data Fig. 7 CellREADR targeting of PNFezf2 and PNCtip2 types in mouse cortex with ADAR2 overexpression.
a–b, Genomic structures of mouse Fezf2 (a) and Ctip2 (b) genes with locations of sesRNAs as indicated, respectively. c, Schematic of binary AAV vectors for targeting neuron types. In READRFezf2-tTA2, a hSyn promoter drives expression of ADAR2 followed by sequences coding for sesRNAFezf2(1), T2a, and tTA2 effector. In TRE3g-mRuby3, the TRE3g promoter drives mRuby3 in response to tTA2 from the READR virus. d, Image of coronal section from a Fezf2-CreER;LoxpSTOPLoxp-H2bGFP mouse brain, showing the distribution pattern of Fezf2+ PNs in S1 somatosensory cortex (d1). Co-injection of AAVs READRFezf2(1)-tTA2 and TRE3g-mRuby3 specifically labeled PNs in L5b and L6 (d2). Co-labeling by CellREADR AAVs and H2bGFP (d3) with magnified view in (d4). Arrows indicate co-labeled cells; arrowhead shows a neuron labeled by CellREADR AAVs but not by Fezf2-H2bGFP (d4). e, Specificity of READRFezf2(1)-tTA2. f, Coronal section of WT brain immuno-stained with a CTIP2 antibody, showing the distribution pattern of Ctip2+ PNs in S1 cortex (f1). Co-injection of AAVs READRCtip2(1) and TRE3g-mRuby3 specifically labeled PNs in L5b (f2). Co-labeling by CellREADR AAVs and CTIP2 antibody (f3) with magnified view in (f4). Arrows show the co-labeled cells; arrowhead showed a mis-labeled cells by READRCtip2(1) (f4). g, Specificity of READRCtip2(1). h, Axonal projection pattern of AAV READRCtip2(1)-tTA2 and TRE3g-eYFP infected PNs in S1 cortex. Representative images showing projections to striatum (h1), thalamus (h2), midbrain (h3), pons (h4) and medulla (h5) (arrows). i, Schematic locations of coronal sections are shown on the right panel. The bars in e and g are mean values, n = 2 mice performed.
Extended Data Fig. 8 CellREADR targeting of additional cortical neuron types in the mouse.
a–b, Expression level and laminar distribution of cortical cell type markers in mice. a, Group plot of selected genes in transcriptomic cell type clusters, based on dataset from the Allen Institute for Brain Science. Gene expression level and cortical distribution were shown. b, Gene expression level of several major cell type marker genes. Each dot indicates the marker gene RNA expression level in cell type cluster. Error bars are mean values ± s.e.m. (n = 8 for Fezf2, n = 8 for Ctip2, n = 28 for SatB2, n = 15 for PlxnD1, n = 7 for Tle4, n = 7 for Foxp2, n = 13 for Rorb, n = 60 for vGAT). PT (pyramidal tract), IT(intratelencephalic) and CT (Corticothalamic) indicate three main excitatory cortical neuronal types. The plots were generated from mouse cortical scRNAseq online tool (https://celltypes.brain-map.org/rnaseq/mouse/v1-alm) with published scRNAseq data. c, Genomic structures of the mouse Satb2 gene with location of a sesRNA as indicated (top). Bottom, Cell labeling pattern in S1 by co-injection of binary vectors described in Fig. 4i. AAVs READRSatb2 and TRE3g-mNeon labeled cells in both upper and deep layers (d1). Satb2 mRNA in-situ hybridization (d2). Co-labeling by READRSatb2 and Satb2 mRNA (d3). e, Magnified view of boxed region in d3. Arrowheads indicate co-labeled cells. f, Satb2 mRNA expression pattern in S1 cortex at P56 from the Allen Mouse Brain Atlas. g, Specificity of READRSatb2 measured as the percent of Satb2+ cells among mNeon cells. h, Genomic structures of the mouse PlxnD1 gene with location of a sesRNA as indicated. i–j, AAVs READRPlxnD1 and TRE3g-mNeon labeled cells in upper layers and L5a in S1 (i1). PlxnD1 mRNA in-situ hybridization (i2). Co-labeling by READRPlxnD1 AAVs and PlxnD1 mRNA (i3). j, Magnified view of boxed region in i3. Arrowheads show the co-labeled cells. k, PlxnD1 mRNA expression in P56 S1 cortex from the Allen Mouse Brain Atlas. l, Specificity of READRPlxnD1 measured as the percent of PlxnD1+ cells among mNeon cells. m, Genomic structures of the mouse Rorb gene with location of a sesRNA. n, AAVs READRRorb and TRE3g-mNeon labeled cells in layer 4 (n1, n3). DAPI staining indicated laminar structure (n2). mNeon labeling pattern is consistent with Rorb mRNA expression in P56 S1 cortex from the Allen Mouse Brain Atlas (o). p, Genomic structures of mouse the vGAT gene with location of a sesRNA as indicated. q-r, binary READRvGAT and TRE3g-mNeon labeled cells (q1). vGAT mRNA in-situ hybridization. (q2). Co-labeling by READRvGAT AAVs and vGAT mRNA (q3). r, Magnified view of rectangle in (q3). Arrowheads show the co-labeled cells. s, vGAT mRNA expression in P56 S1 cortex from the Allen Mouse Brain Atlas. t, Specificity of READRvGAT measured as the percent of vGAT+ cells among mNeon cells. Each bar in g, l and t is the value from one mouse performed (n = 1).
Extended Data Fig. 9 Assessment of cortical cellular immune responses following long-term expression of CellREADR vectors.
a, Schematic of evaluation of the long-term effects of CellREADR in vivo. For each mouse, READRCtip2(3) or CAG-tdT control AAVs were injected into S1 cortex and incubate for three months. Fresh brains were dissected and small pieces of cortical tissue at the injection site were collected. Quantitative PCR was performed immediately. S1 tissues of mice without viral injection were used as control. b, RNA expression level changes of nine genes implicated in glia activation and immunogenicity. Error bars are mean values ± s.e.m. n = 3 biological replicates in different mice performed. Unpaired two-tailed Student’s t-test was used for analysis and P values were given.
Extended Data Fig. 10 Axonal projection pattern of L5/6 CFPNs in caudal forelimb motor area targeted by AAVs READRCtip2 and TRE3g-ChRger2-eYFP.
a, Schematic of binary AAV vectors of READRCtip2(3)-smFlag/tTA2 and TRE3g-ChRger2-eYFP for optogenetic activation and axonal projection tracing. b, Axonal projection pattern of CFPNs infected in CFA. Representative images showing projections to striatum (b2), thalamus (b3, b4), pons (b5) and medulla (b6, arrows). c, Schematic locations of coronal sections in b are shown.
Extended Data Fig. 11 CellREADR targeting of neuron types in rat.
a, Schematic of binary AAV vectors for cell type targeting in rat. In READR vector, a hSyn promoter drives expression of mCherry followed by sequences coding for sesRNA, smFlag and tTA2 effectors. Along with READR, a Reporter vector drives mNeonG expression from a TRE3g promoter in response to tTA2 from the READR vector. b, Genomic structures of the rat vGAT gene with location of a sesRNA as indicated. c, AAVs READRvGAT and TRE3g-mNeon were injected into cortical deep layer and hippocampus. Binary vectors labeled cells shown in cortex (c1). c2, Magnified view of boxed region in c1. vGAT mRNAs were labeled by in-situ hybridization (c3). Co-labeling by mNeon and vGAT mRNA (c4). Arrows showed the co-labeled cells. d, Cell labeling pattern in the hippocampus CA1 region by co-injection of AAVs READRvGAT and TRE3g-mNeon. e, Magnified view of boxed region in d. Arrows indicate co-labeled cells. f–g, Specificity of rat READRvGAT in rat cortex (f) and hippocampus (g) measured as the percent of vGAT+ cells among mNeon cells. h, Genomic structures of the rat Tle4 gene with the location of a sesRNA as indicated. i–j, AAVs READRTle4 and TRE3g-mNeon co-injected into the rat motor cortex labeled cells concentrated in deep layers (i1). Tle4+ PNs were labeled by TLE4 antibody staining (i2, 3). j, Magnified view of the boxed region in i. Arrows indicate co-labeled cells by CellREADR and TLE4 antibody. k, Specificity of rat READRTle4 in rat cortex, measured as the percent of TLE4 + cells among mNeon cells. Each bar in f, g and k is the value from one rat performed (n = 1).
Extended Data Fig. 12 CellREADR vector targeting of neuron types in human cortical ex vivo tissues.
a, Schematic of organotypic platform for of human cortical ex vivo tissues. Left panel was adapted from “Brain (lateral view)”, by BioRender.com (2022). Retrieved from https://app.biorender.com/biorender-templates. b, Schematic of a hSyn-eGFP viral construct used to drive widespread neuronal cell labeling. c. AAVrg-hSyn-eGFP labeled cells were distributed across all layers, and exhibited diverse morphologies (c1). Insets from c1 (c2) and c2 (c3, 4) depict numerous cells with pyramidal morphologies, including prominent vertically oriented apical dendrites. d, FOXP2 expression in human neocortex. FOXP2 mRNA expression pattern taken from the Allen Institute human brain-map (specimen# 4312), showing upper and deep layer expression (arrows) (d1). FOXP2 immunostaining in the current study (magenta) also demonstrated both upper and deep labeling (d2, 4). NeuN immunostaining (red) depicting cortical neurons (d3, 4). Dashed lines delineate pia and white matter. e, READRFOXP2(1) labeling in an organotypic slice derived from the same tissue used in d. Overview of bright field and mNeon native fluorescence in the organotypic slice demonstrating highly restricted labeling, as compared to that observed in d. Inset from e2 (e3) depicting morphologies of upper layer pyramidal neurons. f, Schematics of two singular vectors of READRFOXP2. In READRFOXP2(1), the hSyn promoter drives an expression cassette encoding ClipF, sesRNA1, smV5, and tTA2. In READRFOXP2(2), the hSyn promoter drives an expression cassette encoding ClipF, sesRNA2, smFlag, and FlpO. g–h, Seven days after application of READRFOXP2(2) AAV on DIV 1, tissue was fixed and stained with antibodies against FOXP2 and FLAG (g). h, Boxed region from g. FLAG-labeled cells from READRFOXP2(2) exhibited relatively small somata with short apical dendrites (h1–h3, arrowheads). Non-specific background fluorescence signals (e.g. a blood vessel-like profile) are indicated by thin arrows (h2). i, Quantification of CellREADR specificity measured as the percentage of V5+ cells (for READRFOXP2(1)) and FLAG+ cells (for READRFOXP2(2)) labeled by FOXP2 immunostaining, respectively. Each bar in i is the value from one human brain sample performed (n = 1).
Supplementary information
Supplementary Information
This file contains Supplementary Figs. 1 and 2 and Tables 1–3.
Supplementary Video 1
Optogenetic activation of mouse injected with binary CellREADR viral vectors. Optogenetic activation (CFA, 0.5 s) in a mouse injected with binary READRCtip2/3/ReporterChRger2-eYFP AAVs induces a stepping forelimb movement. The upward movement involves sequentially, elbow, wrist, and digit flexion followed by extension.
Supplementary Video 2
Optogenetic activation of mouse injected with binary PT enhancer viral vectors. Optogenetic activation (CFA, 0.5 s) in a mouse injected with PT-enhancer/ReporterChRger2-eYFP AAVs induces elbow extension accompanied by digit extension.
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Qian, Y., Li, J., Zhao, S. et al. Programmable RNA sensing for cell monitoring and manipulation. Nature 610, 713–721 (2022). https://doi.org/10.1038/s41586-022-05280-1
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DOI: https://doi.org/10.1038/s41586-022-05280-1
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