Fig 1.
ORANGE: An easy-to-implement toolbox for endogenous tagging of proteins in neurons.
(A) Overview of the pORANGE knock-in construct. To clone knock-in constructs, first a 20-bp target sequence for the genomic locus of interest is ligated in the guide RNA cassette. Then, the donor sequence containing the tag of interest is generated by PCR and inserted in the donor DNA cassette. (B) Mechanism of ORANGE-mediated gene targeting. (C) Examples of knock-in neurons expressing GluA1 tagged with GFP, HaloTag, small epitope tags (2× HA, 2× FLAG), or GFP-P2A-Cre recombinase. Dashed boxes indicate zooms. Scale bars, 40 μm for the GluA1-GFP overview (far left), 10 μm for individual overviews, and 5 μm for the zooms. β-act, β-actin; CMV, cytomegalovirus; GFP, green fluorescent protein; GluA, Glutamate receptor AMPA 1; HA, hemagglutinin; NHEJ, nonhomologous end joining; ORANGE, Open Resource for the Application of Neuronal Genome Editing; PAM, protospacer adjacent motif; SpCas9, Streptococcus pyrogenes Cas9; T, target sequence; Term, termination sequence.
Fig 2.
A versatile ORANGE knock-in library for endogenous tagging of proteins in neurons.
(A) Example at low magnification showing four GFP-β-actin knock-in neurons (DIV 21). Zooms show an axon and dendrite, respectively. Scale bars: overview, 200 μm; zoom, 5 μm. (B) Example of two PSD95-Halo knock-in neurons (DIV 21). Zoom shows a single dendrite. Scale bars: overview, 40 μm; zoom, 5 μm. (C) Example of GFP-GluN2b knock-in neuron (DIV 21). Scale bars: overview, 40 μm; zoom, 5 μm. (D) Representative images of ORANGE knock-in neurons, categorized according to protein function or subcellular localization. Neurons were transfected at DIV 3 and imaged at DIV 21. Scale bars, 5 μm. Asterisk indicates signal enhancement with anti-GFP staining (Alexa488 or Alexa647). Arpc5, actin-related protein 2/3 complex subunit 5; CaMKIIα, Calcium/calmodulin-dependent protein kinase type II subunit alpha; CAPS1, Calcium-dependent activator protein for secretion 1; CaV, voltage-dependent Ca2+-channel; DIV, day in vitro; Doc2a, double C2-like domain-containing protein a; FRRS1L, Ferric-chelate reductase 1-like protein; GFP, green fluorescent protein; GluA, glutamate receptor AMPA 1; GluN, Glutamate receptor NMDA 1; GSG1L, Germ cell-specific gene 1-like protein; LC, light chain; Munc13, mammalian uncoordinated 13; Nlgn3, neuroligin 3; ORANGE, Open Resource for the Application of Neuronal Genome Editing; PSD95, postsynaptic protein 95; Rab11, ras-related protein 11; RIM: Rab3-interacting molecule; Shank, SH3 and multiple ankyrin repeat domains protein; Syt7, Synaptotagmin 7; TARP, Transmembrane AMPAR regulatory protein; WASP1, Wiskott-Aldrich syndrome protein 1.
Fig 3.
ORANGE mediates in vivo genome editing.
(A) Overview of ORANGE AAV plasmid. (B) Workflow and time line for in vivo genome editing. (C and D) Confocal images of acute slices from SpCas9 mouse hippocampus injected with PSD95-Halo knock-in (C) and GluA1-Halo knock-in (D) AAV vectors visualized with Halo-JF646 ligand (green). Infected cells are positive for mCherry-KASH (magenta). Scale bar, 100 μm. (E and G) Zooms for acute slices as shown in (C) and (D), respectively. Scale bar, 40 μm. (F and H) Representative images of confocal and gSTED microscopy in acute slices. Shown are dendrites positive for PSD95-Halo (F) and GluA1-Halo knock-in (H). Scale bar, 2 μm. AAV, adeno-associated virus; EF1α, elongation factor 1α; GluA, Glutamate receptor AMPA; gSTED, gated stimulated-emission depletion; ITR, inverted terminal repeat; JF646, Janelia Fluor 646; KASH, Klarsicht, ANC-1, Syne Homology; ORANGE, Open Resource for the Application of Neuronal Genome Editing; pA, polyadenylation; PSD95, postsynaptic protein 95; SO, stratum oriens; SP, stratum pyramidale; SpCas9, S. pyrogenes Cas9; SR, stratum radiatum; T, target sequence.
Fig 4.
Validation of ORANGE labeling efficiency.
(A) Representative images of dendrites transfected with soluble GFP, PSD95-GFP knock-in (KI) construct, or a PSD95-GFP overexpression construct (green) stained with anti-PSD95 (magenta, Alexa568). DIV 21. Scale bar, 5 μm. (B) Correlation between PSD95-GFP KI and anti-PSD95 staining intensity. (C) Quantification of synaptic PSD95 levels, (D) synapse area, and I PSD95 synapse/shaft intensity. (F) Representative images of dendrites coexpressing Homer1c-mCherry (green) and either the empty pORANGE template vector or PSD95-GFP KI construct (blue) stained with anti-PSD95 (magenta, Alexa647). DIV 21. Scale bar, 5 μm. (G) Quantification of PSD95 levels in transfected but KI-negative neurons. Data are represented as means ± SEM. * P < 0.05, **P < 0.01, *** P < 0.001, ANOVA or Student t test. Underlying data can be found in S1 Data. DIV, day in vitro; GFP, green fluorescent protein; HA, hemagglutinin; KI, knock-in; ns, not significant; OE, overexpression; ORANGE, Open Resource for the Application of Neuronal Genome Editing; PSD95, postsynaptic protein 95; RIM1, Rab3-interacting molecule 1.
Fig 5.
Live-cell imaging of intracellular endogenous protein dynamics.
(A) Representative images of dendrites transfected with GFP-β-actin knock-in imaged over time. DMSO or Jasp was added at time point 0. DIV 21. Scale bar, 1 μm. (B) Frame-to-frame correlation of pixel intensity over time for DMSO (green) or Jasp (blue) addition. (C) Quantification of mean frame-to-frame correlation averaged over the last five time points. (D) Representative images of FRAP experiment on dendrites transfected with GFP-β-actin knock-in vector. ROIs were bleached at time point 0 (orange circle). Recovery was followed over time. DIV 21. Scale bar, 1 μm. (E) FRAP analysis of GFP-β-actin knock-in neurons treated with DMSO (control) or Jasp. ROIs were bleached at time point 0 (dotted line). (F) Quantification of mobile fraction calculated from the last five time points of each bleached ROI averaged per neuron. Data are represented as means ± SEM. **P < 0.01, *** P < 0.001, Student t test. Underlying data can be found in S1 Data. DIV day in vitro; FRAP, fluorescence recovery after photobleaching; GFP, green fluorescent protein; Jasp, jasplakinolide; ORANGE, Open Resource for the Application of Neuronal Genome Editing; ROI, region of interest.
Fig 6.
STED microscopy to resolve the subcellular distribution of endogenous proteins in individual neurons.
(A) Representative gSTED image of a GFP-β-actin knock-in neuron (DIV 21) enhanced with anti-GFP (ATTO647N). Scale bar, 20 μm. (B and C) Zooms of axon (B) and dendrite (C) as indicated with boxes in (A) comparing confocal and gSTED imaging. Scale bar, 2 μm; insert scale is 1 μm. (D and E) Line scans from zooms in (B) and (C), respectively. (F) Representative gSTED images of dendrites positive for PSD95-GFP knock-in stained with anti-GFP (green, ATTO647N) and anti-PSD95 staining (magenta, Alexa594). DIV 21. Scale bar, 2 μm. (G) Zooms from (F) of individual synapses resolved with confocal and gSTED. Scale bar, 500 nm. (H) Line scans of confocal and gSTED images shown in (G). (I) Representative gSTED images of dendrites positive for GFP-β-actin knock-in stained with anti-GFP (green, ATTO647N) and anti-PSD95 (magenta, Alexa594). DIV 21. Scale bar, 2 μm. (J) Zooms from (I) of individual spines resolved with confocal and gSTED. Scale bar, 500 nm. (K) Line scans of confocal and gSTED images shown in (J). (L and M) Representative gSTED images of dendrites positive for GSG1L-GFP (L) or FRRS1L-GFP knock-in (M) stained with anti-GFP (green, Alexa488) and anti-PSD95 (magenta, ATTO647N). DIV 21. Scale bar, 5 μm. (N) Representative images of dendrites positive for FRRS1L-GFP knock-in enhanced with anti-GFP (gSTED, green) and coexpressed with tagRFP-ER (confocal, magenta). DIV 21. Scale bar, 2 μm. DIV, day in vitro; ER, endoplasmic reticulum; FRRS1L, Ferric-chelate reductase 1-like protein; GFP, green fluorescent protein; GSG1L, Germ cell-specific gene 1-like protein; gSTED, gated STED; PSD95, postsynaptic protein 95; RFP, red fluorescent protein; STED, stimulated-emission depletion.
Fig 7.
NMDA receptors concentrate in subsynaptic nanodomains and are highly immobilized in synapses.
(A) Representative images of a dendrite positive for GFP-GluN1 KI (green) stained for PSD95 (magenta, Alexa647). Scale bar, 2 μm. (B) Correlation between GFP-GluN1 KI and anti-PSD95 staining intensity within individual GFP-GluN1 puncta. (C) Representative gSTED images of dendrites positive for GFP-GluN1 KI enhanced with anti-GFP (green, ATTO647N) and anti-PSD95 (magenta, Alexa594). DIV 21. Scale bar, 2 μm. (D) Zooms of individual synapses indicated in (C). Scale bar, 500 nm. (E) FWHM analysis of GFP-GluN1 structures comparing width and length of individual synaptic (red) and extrasynaptic (blue) GluN1 clusters. (F) Correlation between GFP-GluN1 cluster area and synapse area (based on anti-PSD95 staining) for individual synapses. (G) Line scan of synapse zoom 3 in (D). (H) Quantification of the number of GFP-GluN1 substructures per synapse. (I) Representative image of dendrite positive for GFP-GluN1 KI stained with anti-GFP (Alexa647). DIV 21. Scale bar, 1 μm. (J) Single-molecule dSTORM reconstruction of example shown in (I). Scale bar, 1 μm. (K) Examples of individual GFP-GluN1 clusters with single localizations plotted and color-coded based on the local density. Scale bar, 200 nm. (L) Quantification of number of GFP-GluN1 nanodomains per cluster. (M) Frequency distribution of GFP-GluN1 nanodomain size. Dotted line indicates nanodomain size cutoff. Bin size: 5 nm. (N) Representative example of GFP-GluN1 (anti-GFP nanobody conjugated to ATTO647N) single-molecule trajectories in a dendrite plotted with a random color and on top of a synapse mask (gray) based on Homer1c-mCherry widefield image. Dotted line indicates cell outline. DIV 21. Scale bar, 1 μm. (O) Zooms of individual spines indicated in (N) with example trajectories of synaptic (red) or extrasynaptic (blue) receptors. Scale bar, 200 nm. (P) Frequency distribution showing the diffusion coefficient of synaptic and extrasynaptic tracks. Data in bar plots are presented as means ± SEM. Underlying data can be found in S1 Data. DIV, day in vitro; dSTORM, direct stochastic optical reconstruction microscopy; FWHM, Full Width at Half Maximum; GFP, green fluorescent protein; GluN, glutamate receptor NMDA; gSTED, gated stimulated-emission depletion; KI, knock-in; NMDA, N-methyl-D-aspartate; PSD95, postsynaptic protein 95.
Fig 8.
Cre-dependent coexpression and labeling of two proteins in single neurons.
(A) Overview of plasmids used for Cre-dependent expression of mCherry-KASH or Synapsin-FLAG in knock-in neurons. (B) Examples of GFP-P2A knock-in–driven expression of mCherry-KASH or Synapsin-FLAG (Alexa568) (magenta) for various knock-ins. DIV 21. Scale bars, 10 μm and 5 μm for the overviews and zooms, respectively. (C) Overview of plasmids used for multiplex knock-in of two proteins in single neurons (ORANGE-CAKE). (D and E) Examples of β3-tubulin-GFP-P2A-Cre (green), Lox GluA1-HA (magenta, Alexa594) double knock-in, (D) and PSD95-GFP-P2A-Cre (green), Lox Halo-β-actin (magenta, JF549) double knock-in (E). Shown are overviews (confocal) and zooms (gSTED). DIV 21. Scale bars, 20 μm for the overviews and 5 μm (dendrites) and 500 nm (spine) for the zooms. (F) Examples of various combinations of GFP-P2A-Cre (green) and Lox (magenta) double knock-ins. HA was visualized by anti-HA staining (Alexa594), and Halo with Halo-JF549 ligand. DIV21. Scale bars, 10 μm and 5 μm for the overviews and zooms, respectively. Asterisk indicates enhancement with anti-GFP antibody (Alexa488). CAKE, conditional activation of knock-in expression; CaV, voltage-dependent Ca2+-channel; DIV, day in vitro; FLEx, flip-excision; GFP, green fluorescent protein; GluA, glutamate receptor AMPA; gRNA, guide RNA; gSTED, gated stimulated-emission depletion; HA, hemagglutinin; hSyn, human Synapsin; JF549, Janelia Fluor 549; KASH, Klarsicht, ANC-1, Syne Homology; ORANGE, Open Resource for the Application of Neuronal Genome Editing; PSD95, postsynaptic protein 95; T, target sequence.