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.
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Molecular Brain Open Access 04 January 2022
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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.
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.
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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.
The authors declare no competing interests.
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
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Integrated supplementary information
Schematic representation of the DNA constructs generated for this study. rAAV constructs were used to produce viruses expressing RecV and the reporter constructs.
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.
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.
(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.
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.
(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.
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.
(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 Figs. 1–11 and Note.
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.
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.
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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
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