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SPARC enables genetic manipulation of precise proportions of cells

Abstract

Many experimental approaches rely on controlling gene expression in select subsets of cells within an individual animal. However, reproducibly targeting transgene expression to specific fractions of a genetically defined cell type is challenging. We developed Sparse Predictive Activity through Recombinase Competition (SPARC), a generalizable toolkit that can express any effector in precise proportions of post-mitotic cells in Drosophila. Using this approach, we demonstrate targeted expression of many effectors in several cell types and apply these tools to calcium imaging of individual neurons and optogenetic manipulation of sparse cell populations in vivo.

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Fig. 1: Schematic description of the SPARC method.
Fig. 2: The SPARC toolkit enables predictable expression of effectors at three levels.
Fig. 3: SPARC2 labels precise proportions of neurons across a diverse set of cell types.
Fig. 4: SPARC2 stochastically labels different subsets of neurons in each animal.
Fig. 5: SPARC enables calcium imaging of single neurons.
Fig. 6: SPARC2 enables optogenetic stimulation of sparse cell populations.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

Code availability

All analysis was carried out using custom-written MATLAB code: https://github.com/wienecke/SPARC. Visual stimuli were programmed with the OpenGL 1.0 API in Visual C#. All code is available on Github and will be made available upon reasonable request from the corresponding author. Source data are provided with this paper.

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Acknowledgements

We thank members of the Clandinin, Wilson and Maimon labs for discussion of the project and manuscript. We thank A. Chakravorty for generating the SPARC-jGCaMP7f plasmids and S. Gratz, K. O’Connor-Giles (Brown University), C. Xie, L. Luo (Stanford University) and B. Pfeiffer and D. Anderson (CalTech) for providing template plasmids for molecular cloning. We also thank G. Rubin and H. Dionne (Janelia Farms) for sharing split-Gal4 stocks and N. Perrimon (Harvard University) for sharing Cas9 stocks. Additionally, stocks obtained from the Bloomington Drosophila Stock Center (National Institutes of Health (NIH) P40OD018537) were used in this study. The project was supported by the NIH (R01EY022638 and 5P30EY026877 to T.R.C. and 5U19NS104655 to T.R.C. and R.E.W). J.I.-B. is an Arnold O. Beckman Postdoctoral Fellow. H.H.Y. is a Howard Hughes Medical Institute (HHMI) fellow of the Jane Coffin Childs Memorial Fund for Medical Research. Y.E.F. is supported by a Hanna H. Grey Fellowship from the HHMI. C.F.R.W. is supported by a National Science Foundation Graduate Research Fellowship (DGE – 1656518). R.I.W. and G.M. are HHMI investigators.

Author information

Authors and Affiliations

Authors

Contributions

J.I.-B., H.H.Y., I.E.W. and T.R.C conceived the study. J.I.-B., C.F.R.W., H.H.Y. and Y.E.F. designed and performed the experiments under the supervision of T.R.C. and R.I.W. J.I.-B., K.C.P., H.H.Y., Y.E.F. and I.G.I. generated, maintained and/or validated transgenic fly stocks under the supervision of T.R.C, R.I.W. and G.M. J.I.-B., C.F.R.W., H.H.Y., Y.E.F. and K.C.P. analyzed the data. J.I.-B. and T.R.C. prepared the manuscript with contributions from C.F.R.W., H.H.Y. and Y.E.F.

Corresponding author

Correspondence to Thomas R. Clandinin.

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The authors declare no competing financial interests.

Additional information

Peer review information Nature Neuroscience thanks Claude Desplan, Olena Riabinina and the other, anonymous, reviewer(s) 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 SPARC development cassettes.

a, b, Schematics of PhiC31-dependent UAS-inversion effector constructs. (a) control construct with canonical attP sites and (b) truncated 34bp_attP experimental construct. c, d”, 34bp_attP-Inversion-GCaMP6f expression (green, c, d) in Mi1 neurons (magenta, c’, d’) counterstained with anti-Bruchpilot (Brp; blue, overlay c”, d”). Fewer Mi1 neurons are labeled at day two post eclosion (cc”) than at day six post eclosion (dd”). e, Schematic of the LexA-OR-Flp expression construct. PhiC31 recombines one of two competing attP target sequences with one attB target sequence to enable either LexA or Flp expression. Reaction 1 leads to LexA expression. Reaction 2 leads to Flp expression. f–f”, Flp-enabled mCD8::GFP expression (green, f) or LexA-driven myr::tdTomato expression in Mi1 neurons (magenta, f’) counterstained with anti-Bruchpilot (Brp; blue, overlay f”). n = 10 optic lobes per genotype. Scale bar: 10 µm.

Extended Data Fig. 2 Plasmid maps and molecular cloning methods for SPARC and SPARC2 constructs.

a, Map of pHD-3xP3-DsRed-ΔattP (a CRISPR-HDR-donor precursor) showing multiple cloning sites for homology arm insertion (right). b, Map of pHD-3xP3-DsRed-ΔattP-CRISPR-donor (example includes homology arms targeting the attP40 region of the Drosophila genome). c, SPARC and SPARC2 cassettes are inserted into pHD-3xP3-DsRed-ΔattP-CRISPR-donor via unique KpnI, NdeI, or BsiWI restriction enzyme sites. SalI restriction enzyme sites in the SPARC2 module allow for one-step swapping of the effector and terminator to generate pHD-SPARC2 donor plasmids. Abbreviations: MCS – multiple cloning site; gRNA – guide RNA; HDVR – hepatitis delta virus ribozyme sequence.

Extended Data Fig. 3 SPARC-GCaMP6f expression in Kenyon cells.

a–d, Anterior view of the Drosophila central brain showing GCaMP6f expression (green) in Kenyon cells (magenta) counterstained with anti-Bruchpilot (Brp; blue). a, SPARC-D-GCaMP6f, no PhiC31. b, SPARC-D-GCaMP6f. c, SPARC-I-GCaMP6f. d, SPARC-S-GCaMP6f. e–h”, GCaMP6f expression (green, eh) in Kenyon cell bodies (magenta, e’h’) with overlay (e”h”). ee”, SPARC-D-GCaMP6f, no PhiC31. GCaMP6f is not detected in Kenyon Cells in the absence of PhiC31. ff”, SPARC-D-GCaMP6f. gg”, SPARC-I-GCaMP6f. hh”, SPARC-S-GCaMP6f. Scale bars: 30 µm (ad), 10 µm (eh”). n > 10 brains per condition from three independent experiments.

Extended Data Fig. 4 SPARC and SPARC2 user guide.

a, Important notes regarding SPARC and SPARC2 use and stock maintenance. b, Example crossing schemes for SPARC or SPARC2 to allow expression of effectors.

Supplementary information

Supplementary Information

Supplementary Tables 5 and 7 and Supplementary Notes on fly genotypes (by figure) and origin of transgenes

Reporting Summary

Supplementary Table

Supplementary Tables 1–4 and 6 describe reagents from the Methods section.

Source data

Source Data Fig. 3

Original cell counts and complete statistical analyses

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Isaacman-Beck, J., Paik, K.C., Wienecke, C.F.R. et al. SPARC enables genetic manipulation of precise proportions of cells. Nat Neurosci 23, 1168–1175 (2020). https://doi.org/10.1038/s41593-020-0668-9

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