RecV recombinase system for in vivo targeted optogenomic modifications of single cells or cell populations

Abstract

Brain circuits comprise vast numbers of interconnected neurons with diverse molecular, anatomical and physiological properties. To allow targeting of individual neurons for structural and functional studies, we created light-inducible site-specific DNA recombinases based on Cre, Dre and Flp (RecVs). RecVs can induce genomic modifications by one-photon or two-photon light induction in vivo. They can produce targeted, sparse and strong labeling of individual neurons by modifying multiple loci within mouse and zebrafish genomes. In combination with other genetic strategies, they allow intersectional targeting of different neuronal classes. In the mouse cortex they enable sparse labeling and whole-brain morphological reconstructions of individual neurons. Furthermore, these enzymes allow single-cell two-photon targeted genetic modifications and can be used in combination with functional optical indicators with minimal interference. In summary, RecVs enable spatiotemporally precise optogenomic modifications that can facilitate detailed single-cell analysis of neural circuits by linking genetic identity, morphology, connectivity and function.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Design of the RecV systems.
Fig. 2: Optogenomic modifications with spatiotemporal and cell-class-specific precision in vivo.
Fig. 3: CreV allows optogenomic modifications in multiple tissues of Danio rerio.
Fig. 4: Cortical PCs labeled RecVs and were reconstructed at the whole-brain level.
Fig. 5: 2P-guided targeted single-cell optogenomic modifications by iCreV in mouse neocortex.

Data availability

DNA sequences of the NCreV, CCreV, NDreV, CDreV, iCreV, iDreV and iFlpV created in this work are curated in National Institute of Health, GenBank. Accession codes are NCreV, MT036266; CCreV, MT036267; NDreV, MT036268; CDreV, MT036269; iCreV, MN944913; iFlpV, MN944914; and iDreV, MN944915. AAV iCreV, 140135; AAV iDreV, 140136; AAV iFlpV, 140137; AAV NCreV, 140131; AAV CCreV, 140132; AAV NDreV, 140134; and AAV CDreV, 140133. Plasmids have been deposited in Addgene with the indicated accession codes. All in vivo and in vitro raw data images used in all figures presented in the paper are available from the corresponding author upon request. Source data files for all figures with graphs are provided in raw tabular form as Excel files.

Code availability

The two-photon microscope was operated using ScanImage v.5.3 (Vidrio Technologies, LLC) software and custom software written in LabView 2015 (National Instruments). The code is available upon request to the authors.

References

  1. 1.

    Herculano-Houzel, S. The human brain in numbers: a linearly scaled-up primate brain. Front. Hum. Neurosci. 3, 31 (2009).

    PubMed  PubMed Central  Google Scholar 

  2. 2.

    Huang, Z. J. & Zeng, H. Genetic approaches to neural circuits in the mouse. Annu. Rev. Neurosci. 36, 183–215 (2013).

    CAS  PubMed  Google Scholar 

  3. 3.

    Nagy, A. Cre recombinase: the universal reagent for genome tailoring. Genesis 26, 99–109 (2000).

    CAS  PubMed  Google Scholar 

  4. 4.

    Branda, C. S. & Dymecki, S. M. Talking about a revolution: the impact of site-specific recombinases on genetic analyses in mice. Dev. Cell 6, 7–28 (2004).

    CAS  PubMed  Google Scholar 

  5. 5.

    Glaser, S., Anastassiadis, K. & Stewart, A. F. Current issues in mouse genome engineering. Nat. Genet. 37, 1187–1193 (2005).

    CAS  PubMed  Google Scholar 

  6. 6.

    Velez-Fort, M. et al. The stimulus selectivity and connectivity of layer six principal cells reveals cortical microcircuits underlying visual processing. Neuron 83, 1431–1443 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Marshel, J. H., Mori, T., Nielsen, K. J. & Callaway, E. M. Targeting single neuronal networks for gene expression and cell labeling in vivo. Neuron 67, 562–574 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Rompani, S. B. et al. Different modes of visual integration in the lateral geniculate nucleus revealed by single-cell-initiated transsynaptic tracing. Neuron 93, 767–776 e766 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Luo, L. Fly MARCM and mouse MADM: genetic methods of labeling and manipulating single neurons. Brain Res. Rev. 55, 220–227 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Denk, W., Strickler, J. H. & Webb, W. W. Two-photon laser scanning fluorescence microscopy. Science 248, 73–76 (1990).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Shimizu-Sato, S., Huq, E., Tepperman, J. M. & Quail, P. H. A light-switchable gene promoter system. Nat. Biotechnol. 20, 1041–1044 (2002).

    CAS  PubMed  Google Scholar 

  12. 12.

    Levskaya, A., Weiner, O. D., Lim, W. A. & Voigt, C. A. Spatiotemporal control of cell signalling using a light-switchable protein interaction. Nature 461, 997–1001 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Kawano, F., Suzuki, H., Furuya, A. & Sato, M. Engineered pairs of distinct photoswitches for optogenetic control of cellular proteins. Nat. Commun. 6, 6256 (2015).

    CAS  PubMed  Google Scholar 

  14. 14.

    Muller, K. et al. A red/far-red light-responsive bi-stable toggle switch to control gene expression in mammalian cells. Nucleic Acids Res. 41, e77 (2013).

    PubMed  PubMed Central  Google Scholar 

  15. 15.

    Lungu, O. I. et al. Designing photoswitchable peptides using the AsLOV2 domain. Chem. Biol. 19, 507–517 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Crefcoeur, R. P., Yin, R., Ulm, R. & Halazonetis, T. D. Ultraviolet-B-mediated induction of protein–protein interactions in mammalian cells. Nat. Commun. 4, 1779 (2013).

    PubMed  Google Scholar 

  17. 17.

    Strickland, D. et al. TULIPs: tunable, light-controlled interacting protein tags for cell biology. Nat. Methods 9, 379–384 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Kennedy, M. J. et al. Rapid blue-light-mediated induction of protein interactions in living cells. Nat. Methods 7, 973–975 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Motta-Mena, L. B. et al. An optogenetic gene expression system with rapid activation and deactivation kinetics. Nat. Chem. Biol. 10, 196–202 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Wang, X., Chen, X. & Yang, Y. Spatiotemporal control of gene expression by a light-switchable transgene system. Nat. Methods 9, 266–269 (2012).

    CAS  PubMed  Google Scholar 

  21. 21.

    Bugaj, L. J., Choksi, A. T., Mesuda, C. K., Kane, R. S. & Schaffer, D. V. Optogenetic protein clustering and signaling activation in mammalian cells. Nat. Methods 10, 249–252 (2013).

    CAS  PubMed  Google Scholar 

  22. 22.

    Nihongaki, Y., Yamamoto, S., Kawano, F., Suzuki, H. & Sato, M. CRISPR-Cas9-based photoactivatable transcription system. Chem. Biol. 22, 169–174 (2015).

    CAS  PubMed  Google Scholar 

  23. 23.

    Lee, S. et al. Reversible protein inactivation by optogenetic trapping in cells. Nat. Methods 11, 633–636 (2014).

    CAS  PubMed  Google Scholar 

  24. 24.

    Dagliyan, O. et al. Engineering extrinsic disorder to control protein activity in living cells. Science 354, 1441–1444 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Gasser, C. et al. Engineering of a red-light-activated human cAMP/cGMP-specific phosphodiesterase. Proc. Natl Acad. Sci. USA 111, 8803–8808 (2014).

    CAS  PubMed  Google Scholar 

  26. 26.

    Wu, Y. I. et al. A genetically encoded photoactivatable Rac controls the motility of living cells. Nature 461, 104–108 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Strickland, D., Moffat, K. & Sosnick, T. R. Light-activated DNA binding in a designed allosteric protein. Proc. Natl Acad. Sci. USA 105, 10709–10714 (2008).

    CAS  PubMed  Google Scholar 

  28. 28.

    Lee, J. et al. Surface sites for engineering allosteric control in proteins. Science 322, 438–442 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Polstein, L. R. & Gersbach, C. A. A light-inducible CRISPR-Cas9 system for control of endogenous gene activation. Nat. Chem. Biol. 11, 198–200 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Konermann, S. et al. Optical control of mammalian endogenous transcription and epigenetic states. Nature 500, 472–476 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Yazawa, M., Sadaghiani, A. M., Hsueh, B. & Dolmetsch, R. E. Induction of protein–protein interactions in live cells using light. Nat. Biotechnol. 27, 941–945 (2009).

    CAS  PubMed  Google Scholar 

  32. 32.

    Taslimi, A. et al. Optimized second-generation CRY2-CIB dimerizers and photoactivatable Cre recombinase. Nat. Chem. Biol. 12, 425–430 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Kawano, F., Okazaki, R., Yazawa, M. & Sato, M. A photoactivatable Cre-loxP recombination system for optogenetic genome engineering. Nat. Chem. Biol. 12, 1059–1064 (2016).

    CAS  PubMed  Google Scholar 

  34. 34.

    Schindler, S. E. et al. Photo-activatable Cre recombinase regulates gene expression in vivo. Sci. Rep. 5, 13627 (2015).

    PubMed  PubMed Central  Google Scholar 

  35. 35.

    Jung, H. et al. Noninvasive optical activation of Flp recombinase for genetic manipulation in deep mouse brain regions. Nat. Commun. 10, 314 (2019).

    PubMed  PubMed Central  Google Scholar 

  36. 36.

    Hochrein, L., Mitchell, L. A., Schulz, K., Messerschmidt, K. & Mueller-Roeber, B. L-SCRaMbLE as a tool for light-controlled Cre-mediated recombination in yeast. Nat. Commun. 9, 1931 (2018).

    PubMed  PubMed Central  Google Scholar 

  37. 37.

    Loros, J. J. & Dunlap, J. C. Genetic and molecular analysis of circadian rhythms in Neurospora. Ann. Rev. Physiol. 63, 757–794 (2001).

    CAS  Google Scholar 

  38. 38.

    Hirrlinger, J. et al. Split-Cre complementation indicates coincident activity of different genes in vivo. PLoS ONE 4, e4286 (2009).

    PubMed  PubMed Central  Google Scholar 

  39. 39.

    Jullien, N., Sampieri, F., Enjalbert, A. & Herman, J. P. Regulation of Cre recombinase by ligand-induced complementation of inactive fragments. Nucleic Acids Res. 31, e131 (2003).

    PubMed  PubMed Central  Google Scholar 

  40. 40.

    Wang, P. et al. Intersectional Cre driver lines generated using split-intein mediated split-Cre reconstitution. Sci. Rep. 2, 497 (2012).

    PubMed  PubMed Central  Google Scholar 

  41. 41.

    Guo, F., Gopaul, D. N. & van Duyne, G. D. Structure of Cre recombinase complexed with DNA in a site-specific recombination synapse. Nature 389, 40–46 (1997).

    CAS  PubMed  Google Scholar 

  42. 42.

    Vaidya, A. T., Chen, C. H., Dunlap, J. C., Loros, J. J. & Crane, B. R. Structure of a light-activated LOV protein dimer that regulates transcription. Sci. Signal. 4, ra50 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Zoltowski, B. D. et al. Conformational switching in the fungal light sensor Vivid. Science 316, 1054–1057 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Cardin, J. A. et al. Driving fast-spiking cells induces gamma rhythm and controls sensory responses. Nature 459, 663–667 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Anastassiadis, K. et al. Dre recombinase, like Cre, is a highly efficient site-specific recombinase in E. coli, mammalian cells and mice. Dis. Model. Mech. 2, 508–515 (2009).

    CAS  PubMed  Google Scholar 

  46. 46.

    Sauer, B. & McDermott, J. DNA recombination with a heterospecific Cre homolog identified from comparison of the pac-c1 regions of P1-related phages. Nucleic Acids Res. 32, 6086–6095 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Andrews, B. J., Proteau, G. A., Beatty, L. G. & Sadowski, P. D. The FLP recombinase of the 2µ circle DNA of yeast: interaction with its target sequences. Cell 40, 795–803 (1985).

    CAS  PubMed  Google Scholar 

  48. 48.

    Chen, Y., Narendra, U., Iype, L. E., Cox, M. M. & Rice, P. A. Crystal structure of a Flp recombinase–Holliday junction complex: assembly of an active oligomer by helix swapping. Mol. Cell 6, 885–897 (2000).

    CAS  PubMed  Google Scholar 

  49. 49.

    Raymond, C. S. & Soriano, P. High-efficiency FLP and PhiC31 site-specific recombination in mammalian cells. PLoS ONE 2, e162 (2007).

    PubMed  PubMed Central  Google Scholar 

  50. 50.

    Chan, K. Y. et al. Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems. Nat. Neurosci. 20, 1172–1179 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51.

    Soriano, P. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat. Genet. 21, 70–71 (1999).

    CAS  PubMed  Google Scholar 

  52. 52.

    Daigle, T. L. et al. A suite of transgenic driver and reporter mouse lines with enhanced brain-cell-type targeting and functionality. Cell. 174, 465–480.e422 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 53.

    Pan, Y. A. et al. Zebrabow: multispectral cell labeling for cell tracing and lineage analysis in zebrafish. Development 140, 2835–2846 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54.

    Gong, H. et al. High-throughput dual-colour precision imaging for brain-wide connectome with cytoarchitectonic landmarks at the cellular level. Nat. Commun. 7, 12142 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. 55.

    Dana, H. et al. High-performance calcium sensors for imaging activity in neuronal populations and microcompartments. Nat. Methods 16, 649–657 (2019).

    CAS  PubMed  Google Scholar 

  56. 56.

    Joyner, A. L. & Zervas, M. Genetic inducible fate mapping in mouse: establishing genetic lineages and defining genetic neuroanatomy in the nervous system. Dev. Dyn. 235, 2376–2385 (2006).

    PubMed  Google Scholar 

  57. 57.

    Dimidschstein, J. et al. A viral strategy for targeting and manipulating interneurons across vertebrate species. Nat. Neurosci. 19, 1743–1749 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. 58.

    Liu, Y. J. et al. Tracing inputs to inhibitory or excitatory neurons of mouse and cat visual cortex with a targeted rabies virus. Curr. Biol. 23, 1746–1755 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. 59.

    Tervo, D. G. et al. A designer AAV variant permits efficient retrograde access to projection neurons. Neuron 92, 372–382 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. 60.

    Cetin, A., Komai, S., Eliava, M., Seeburg, P. H. & Osten, P. Stereotaxic gene delivery in the rodent brain. Nat. Protoc. 1, 3166–3173 (2006).

    CAS  PubMed  Google Scholar 

  61. 61.

    Franklin, K. B. J. & Paxinos, G. The Mouse Brain in Stereotaxic Coordinates 18th edn (Academic Press, 1997).

  62. 62.

    Cho, J. R. et al. Dorsal Raphe dopamine neurons modulate arousal and promote wakefulness by salient stimuli. Neuron 94, 1205–1219.e1208 (2017).

    CAS  PubMed  Google Scholar 

  63. 63.

    Gang, Y. et al. Embedding and chemical reactivation of green fluorescent protein in the whole mouse brain for optical micro-imaging. Front. Neurosci. 11, 121 (2017).

    PubMed  PubMed Central  Google Scholar 

  64. 64.

    Li, A. et al. Micro-optical sectioning tomography to obtain a high-resolution atlas of the mouse brain. Science 330, 1404–1408 (2010).

    CAS  PubMed  Google Scholar 

  65. 65.

    Pologruto, T. A., Sabatini, B. L. & Svoboda, K. ScanImage: flexible software for operating laser scanning microscopes. Biomed. Eng. Online 2, 13 (2003).

    PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We are grateful to the Structured Science teams at the Allen Institute for technical support with stereotaxic injections and mouse colony management. The work was funded by the Allen Institute for Brain Science; NIMH BRAIN Initiative grant no. RF1MH114106 to A.Cetin; the NSFC Science Fund for Creative Research Group of China (grant 61721092) to H.G., Q.L. and S.Z.; NIH Brain Initiative grant no. RF1MH117069 to V.G.; the Colvin divisional fellowship of the Division of Biology and Biological Engineering, California Institute of Technology, to A.K.; and NIH BRAIN Initiative grant U01NS107610 to M.S. The creation of the Ai139 mouse line was supported by NIH grant no. R01DA036909 to B.T. We thank S. Durdu, H. Bayer, D. Schrom, B. Kerman and K. Yonehara for critical reading and feedback. We thank the Allen Institute founder, P.G. Allen, for his vision, encouragement and support.

Author information

Affiliations

Authors

Contributions

A. Cetin conceptualized the light-inducible recombinase system. S.Y. performed cloning and characterization of the constructs and participated in image acquisition. B.O. and P.B. performed surgeries, immunohistochemistry and image acquisition. T.Z. Performed cloning. M.M. performed some of the surgeries and light stimulations. T.L.D. performed some of the initial cloning experiments. B.T. and H.Z. contributed to the generation of the Ai139 transgenic mice. H.G., Q.L. and S.Z. acquired fMOST data. X.K. and Y.W. performed Neurolucida reconstructions. V.G. and A.K. designed deep brain imaging experiments and generated the associated data, figure and text. A.K. performed deep brain imaging experiments. S.C. and P.B. performed 2P-induced recombination experiments. A. Curtright and A.D. performed zebrafish experiments. R.C., P.Y. and M.S. performed targeted single-cell 2P experiments and combinatorial cortical jGCaMP7F calcium imaging experiments. A. Cetin and H.Z. designed and coordinated the study, and wrote the manuscript with inputs from all co-authors.

Corresponding author

Correspondence to Ali Cetin.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nina Vogt was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team

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

Integrated supplementary information

Supplementary Figure 1

Schematic representation of the DNA constructs generated for this study. rAAV constructs were used to produce viruses expressing RecV and the reporter constructs.

Supplementary Figure 2 In vitro two-photon stimulation of DreV.

Mammalian cells were co- transfected with EF1a-EGFP, EF1a Dre red fluorescence reporter, EF1a-NDreV, and EF1a-CDreV plasmids. Two-photon activation was conducted at 48 hrs after transfection at various conditions, and reporter expression was observed 36 hrs post stimulation. Two-photon activation conditions were as follows: λ = 900 nm, 90 mW, 1 ms/line (512 lines), 200 µm x 200 µm scan area, and duration of: 1) 3 mins (data not shown due to no signal), 2) 6 mins, 3) 9 mins, 4) 12 mins, 5) 12 mins (repeat), 6) 15 mins. In condition 7 randomly selected single cells were scanned in 5 areas, 36 x 36 µm each, separated by 40 µm roughly in a straight line across the plate with a duration of 1–10 seconds per area. The experiments were independently repeated twice with similar results. Top left scale bar: 100 µm.

Supplementary Figure 3 In vitro efficiency comparisons of optogenomic modification constructs used in this study.

(a) Relative fluorescence intensity of Cre dependent and Dre dependent fluorescent reporters 48 hrs after 20 minutes of light induction for different co-expression constructs. The N- and the C-termini of the CreV recombinase were combined using a variety of approaches. In the first construct, NCreV and CCreV constructs were mixed together. Constructs 2,3 and 4 contain NCre linked with VVD and CCre all within the same open reading frame, with or without a 5 glycine linker. Constructs 5 and 6 contain both NCreV and CCreV linked by the ribosome skipping peptide PQR. Constructs 7 and 8 have NCreV and CCreV linked by IRES sequence instead of PQR. Construct 11 is the DreV version of the most successful CreV co-expression construct. Controls are represented by reporters alone. (b) Comparison of the light-inducible recombination mediated by improved CreV and DreV. Cells were co-transfected with the appropriate Cre or Dre reporters, and recombinase constructs 1. NCreV and CCreV, 2. Cre-Magnets 3. iCreV, 4. NDreV and CDreV, 5. Dre-Magnets 6. iDreV. Images were taken 48 hours after 20 minutes of light induction. (c) Comparison of iCreV with improved CRY2 based light-inducible Cre recombination system. Cells were transiently transfected with fluorescent reporter along with either iCreV or improved CRY2/CIB1 based constructs. 48 hours after various durations of light stimulation average fluorescence values were quantified with 4 replicas per condition. Each experiment is represented by 4 replicas. The line across the box represents the median, the lower and upper hinges correspond to the 25th and 75th percentiles, and the upper and lower whiskers extend from the hinge to the largest or smallest values no further than 1.5 * inter-quartile range (IQR) from the hinge. Source data

Supplementary Figure 4 In vivo testing and comparison of iCreV with Cre-Magnets.

Ai14 tdTomato reporter mice received RO injection of PHP.eB- iCreV and Cre-Magnet viruses along with EF1a-eGFP control virus (n=2 per case). For the indicated groups light stimulation was applied on the left hemisphere two weeks after virus injection. (a) tdTomato expression in Ai14 mice injected with PHP.eB-Cre-Magnets. 4466 (left) and 4244 (right) cells per section (CPS) were labeled with light stimulation, and 43 (left) and 60 (right) CPS were labeled in the absence of light. Labeled cells in the absence of light are indicated by white arrows. (b) tdTomato expression in Ai14 mice injected with PHP.eB-iCreV. 2858 (left) and 4446 (right) CPS were labeled with light stimulation, and no labeling was found in the absence of light. Scale bars: Overviews 1 mm, smaller brain segments 200 µm.

Supplementary Figure 5 Generation of a light-inducible Flp recombinase.

(a) Schematic of iFlpV constructs and sequence of FlpO. Split sites are indicated with arrows and the construct number. (b) Relative fluorescence intensity after transfection of iFlpV and reporter constructs, measured 48 hours after illumination for 20 minutes. The line across the box represents the median, the lower and upper hinges correspond to the 25th and 75th percentiles, and the upper and lower whiskers extend from the hinge to the largest or smallest values no further than 1.5 * inter-quartile range (IQR) from the hinge. (c) Heatmap of fluorescence intensity after transfection of iFlpV variants and reporter constructs measured 48 hours after 20 minutes of light stimulation. The 62 iFlpV variants, iFlpV and negative control were arranged in an 8x8 matrix. The last two brightest conditions are iFlpV2 (aa 27) with an additional linker sequence and iFlpV2 as a control. Source data

Supplementary Figure 6 RecV viruses allow efficient light-mediated optogenomic modifications at different loci.

Various reporter mice (n = 2 per case) received RO injection of the indicated PHP.eB rAAVs. Light stimulation for indicated groups was conducted on the left hemisphere two weeks post injection. (a) Light-induced nuclear-localized tdTomato expression in Ai75 mice (from the Rosa26 locus) injected with AAV-PHP.eB EF1a-Cre virus with 72669 (left) and 172606 (right) cells per slice (CPS). (b) Light-induced nuclear-localized tdTomato expression in Ai75 mice injected with AAV-PHP.eB EF1a-iCreV virus with 9572 (left) and 14211 (right) CPS. (c) Nuclear-localized tdTomato expression in Ai75 mice injected with AAV-PHP.eB EF1a-iCreV virus without light stimulation with 1 (left) and 0 (right) CPS. (d) Light-induced ChrimsonR expression in Ai167 mice (from the TIGRE locus33) injected with AAV-PHP.eB EF1a-iCreV with 161 (left) and 2257 (right) CPS. Two coronal planes are shown for each injection (top row) with enlarged views (lower two rows) for areas indicated by the red boxes. Scale bars: Overviews 1 mm, smaller brain segments 200 µm.

Supplementary Figure 7 Localized iCreV-mediated GCaMP6s expression and optical physiology within striatum.

(a) Virus application scheme and implant for one-photon illumination and GCaMP6s recordings. GCaMP6s reporter mice were locally injected with 1:1 mixture of PHP.eB.iCreV and AAV5.CAG.tdTomato and implanted with 400 μm optical fiber. After 7 days, fiber photometry signal was recorded as baseline activity, and 1P illumination was performed by a 447nm laser using a 200 µm fiber, 5mW, 100ms pulses, 1Hz for 30 minutes. At day 14 fiber photometry signal was measured again. (b) tdTomato expression and GCaMP6s expression as observed under the fiber tip after iCreV activation. (c) Fiber photometry activity in the striatum. ‘Before’ represents baseline activity before illumination -session 2, ‘after’ represents GCaMP6s activity a week after illumination -session 3. (d) Plots of ΔF/F over time and area, before and after illumination (n= 2 mice, 1 trial each).

Supplementary Figure 8 Lower dose of CreV viruses and shorter duration of light induction leads to sparse and strong labeling of individual neurons.

Ai139 Cre-dependent EGFP reporter mice were injected with the 1:1 mixture of NCreV and CCreV rAAVs in the visual or somatosensory cortex, followed by indicated durations of light stimulation two weeks after injection. (a) EGFP expression in Ai139 mice injected with undiluted viruses, and with 30 minutes of light exposure; (b) EGFP expression in Ai139 mice injected with undiluted viruses, and with 5 minutes of light; (c) EGFP expression in Ai139 mice injected with undiluted viruses, and with 3 minutes of light; (d) EGFP expression in Ai139 mice injected with 1:9 dilution of viral solution, and with 5 minutes of light. Images are maximum projections of 100 consecutive fMOST images (each 1 µm-thick). Each condition was repeated in two mice, and fMOST images were obtained for one mouse per group. Scale bars: 200 μm.

Supplementary Figure 9 In vivo two-photon stimulation and sparse recombination using iCreV.

(a) Two-photon stimulated EGFP expression in Ai139 mice. Mice received stereotaxic injections of a 1:5 mixture of EF1a-iCreV:EF1a-tdTomato into VISp, followed by 2P stimulation two to three weeks post-injection. Discrete 400μm x 400μm regions of layer 2/3, approximately 150–250μm below the pial surface (gray boxes), were stimulated at λ = 910nm for 15 minutes each. Two weeks later, animals were perfused. Scale bar is 1mm. (b) High magnification images of eGFP-filled neurons within approximate stimulated regions in (a). Scale bar is 50μm. (c) Individual neuronal processes (right panels) and terminations (left panels) in multiple subcortical regions. Scale bars are 10μm (left) and 30μm (right). Experiments were repeated in two mice with similar results.

Supplementary Figure 10 Additional examples of the in vivo single-cell 2P-induced labeling.

Additional two examples of the 2P induction experiment (15mW, 10min in a and 15mW, 3min in b), following the same preparation and experiment protocol in Fig. 5b. tdTomato reporter Ai14 mice were locally injected with a mixture of rAAV iCreV and rAAV EGFP viruses. Arrows indicate the target cells. Asterisk indicates an induced non-targeted cell. The background red signal observed in the pre-induction session is due to either incomplete shielding of ambient light after surgery or high multiplicity of infection related issues. Experiments were repeated in five mice with similar results. See Supplementary Fig. 11 for background expression rate.

Supplementary Figure 11 Background induction in the mouse cohort for the 2P experiment.

(a) Example images of the first imaging session showed some cells already expressed tdTomato, indicating some background induction prior to two-photon stimulation. tdTomato reporter Ai14 mice were locally injected with a mixture of rAAV iCreV and rAAV EGFP viruses. Arrows indicate the induced cells. (b) Quantification of this background induction rate showed an average of 3% (N = 8 mice). This induction was likely due to incomplete shielding of the ambient light or high multiplicity of infection. (c) Quantification of all the induction plane depths for each field of view associated with the two-photon induction experiment (related to Fig. 5c, d).

Supplementary information

41592_2020_774_MOESM3_ESM.mov

Mouse visual cortical PCs reconstructed at whole-brain level; horizontal axis rotation. Eight EGFP-labeled neurons were imaged by fMOST and reconstructed from barrel cortex of a mouse brain, and are presented in 3D within a partial brain contour composed of serial reconstructed contours of coronal brain sections. The eight PCs include three L2/3 PCs (in pink) having ipsilateral cortico-cortical projections, two L2/3 PCs (in red) having contralateral cortico-cortical projections and three L5 TTPCs (thick-tufted PCs, one in green, one in blue, one in light blue) having cortico-subcortical projections. Note: local axonal clusters are incomplete because the labeling at the region around their somata is too dense for tracing fine axonal branches.

41592_2020_774_MOESM4_ESM.mov

Mouse visual cortical PCs reconstructed at whole-brain level; vertical axis rotation. Eight EGFP-labeled neurons were imaged by fMOST and reconstructed from barrel cortex of a mouse brain, and are presented in 3D within a partial brain contour composed of serial reconstructed contours of coronal brain sections. The eight PCs include three L2/3 PCs (in pink) having ipsilateral cortico-cortical projections, two L2/3 PCs (in red) having contralateral cortico-cortical projections and three L5 TTPCs (thick-tufted PCs, one in green, one in blue, one in light blue) having cortico-subcortical projections. Note: local axonal clusters are incomplete because the labeling at the region around their somata is too dense for tracing fine axonal branches.

Supplementary Information

Supplementary Figs. 1–11 and Note.

Reporting Summary

Supplementary Video 1

Mouse visual cortical PCs reconstructed at whole-brain level; horizontal axis rotation. Eight EGFP-labeled neurons were imaged by fMOST and reconstructed from barrel cortex of a mouse brain, and are presented in 3D within a partial brain contour composed of serial reconstructed contours of coronal brain sections. The eight PCs include three L2/3 PCs (in pink) having ipsilateral cortico-cortical projections, two L2/3 PCs (in red) having contralateral cortico-cortical projections and three L5 TTPCs (thick-tufted PCs, one in green, one in blue, one in light blue) having cortico-subcortical projections. Note: local axonal clusters are incomplete because the labeling at the region around their somata is too dense for tracing fine axonal branches.

Supplementary Video 2

Mouse visual cortical PCs reconstructed at whole-brain level; vertical axis rotation. Eight EGFP-labeled neurons were imaged by fMOST and reconstructed from barrel cortex of a mouse brain, and are presented in 3D within a partial brain contour composed of serial reconstructed contours of coronal brain sections. The eight PCs include three L2/3 PCs (in pink) having ipsilateral cortico-cortical projections, two L2/3 PCs (in red) having contralateral cortico-cortical projections and three L5 TTPCs (thick-tufted PCs, one in green, one in blue, one in light blue) having cortico-subcortical projections. Note: local axonal clusters are incomplete because the labeling at the region around their somata is too dense for tracing fine axonal branches.

Source data

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Yao, S., Yuan, P., Ouellette, B. et al. RecV recombinase system for in vivo targeted optogenomic modifications of single cells or cell populations. Nat Methods 17, 422–429 (2020). https://doi.org/10.1038/s41592-020-0774-3

Download citation