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. 2020 Sep 9;107(5):836-853.e11.
doi: 10.1016/j.neuron.2020.06.003. Epub 2020 Jun 22.

Comprehensive Dual- and Triple-Feature Intersectional Single-Vector Delivery of Diverse Functional Payloads to Cells of Behaving Mammals

Lief E Fenno  1 , Charu Ramakrishnan  2 , Yoon Seok Kim  2 , Kathryn E Evans  2 , Maisie Lo  2 , Sam Vesuna  2 , Masatoshi Inoue  2 , Kathy Y M Cheung  2 , Elle Yuen  2 , Nandini Pichamoorthy  2 , Alice S O Hong  2 , Karl Deisseroth  3
Affiliations

Comprehensive Dual- and Triple-Feature Intersectional Single-Vector Delivery of Diverse Functional Payloads to Cells of Behaving Mammals

Lief E Fenno et al. Neuron. .

Abstract

The resolution and dimensionality with which biologists can characterize cell types have expanded dramatically in recent years, and intersectional consideration of such features (e.g., multiple gene expression and anatomical parameters) is increasingly understood to be essential. At the same time, genetically targeted technology for writing in and reading out activity patterns for cells in living organisms has enabled causal investigation in physiology and behavior; however, cell-type-specific delivery of these tools (including microbial opsins for optogenetics and genetically encoded Ca2+ indicators) has thus far fallen short of versatile targeting to cells jointly defined by many individually selected features. Here, we develop a comprehensive intersectional targeting toolbox including 39 novel vectors for joint-feature-targeted delivery of 13 molecular payloads (including opsins, indicators, and fluorophores), systematic approaches for development and optimization of new intersectional tools, hardware for in vivo monitoring of expression dynamics, and the first versatile single-virus tools (Triplesect) that enable targeting of triply defined cell types.

Keywords: Cre; Flp; VCre; calcium imaging; channelrhodopsin; fluorophores; intersectional; optogenetics; recombinase; targeting.

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Conflict of interest statement

Declaration of Interests The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. INTRSECT strategy and engineering pipeline.
A,D) INTRSECT molecular designs for single open reading frame (‘ORF’; A) and double ORF (D) in three boolean configurations (Cre AND Flp, Cre AND NOT Flp, Flp AND NOT Cre). Reagents for each configuration are listed. B,E) Activity of Cre and Flp to move the single ORF (B) and double ORF (E) INTRSECT starting configurations (top) to the active (dotted box, middle), and inactivated (bottom) states. C,F) From top to bottom, how initial DNA configuration for single ORF (C) and double ORF (F) constructs transition to the active state after recombinase-dependent rearrangement, mRNA processing that removes introns containing recombinase recognition sites, and translation without addition of extraneous sequence (crystal structure in C: GCaMP6m PDB: 3wld (Ding et al., 2014); F: iC++ PDB: 6csn (Kato et al., 2018)). G) Standardized engineering pipeline for production of novel INTRSECT constructs consisting of (left to right) design of intron placement and cloning, RT-PCR to ensure proper splicing, flow cytometry to assay proper expression, and functional testing (in cultured neurons or HEK cells) to compare with the parent. H) Electrophysiology in cultured neurons expressing WT, Con/Fon, Con/Foff, or Coff/Fon variants of ChRmine 3.3-p2a-oScarlet (left) or iC++-EYFP (right) with recombinases. See Figs. s1–s4.
Figure 2.
Figure 2.. Standardized INTRSECT design/implementation.
A) RT-PCR testing and mis-splicing resolution approach for new INTRSECT constructs. B,E) Mis-spliced RT-PCR results for INTRSECT bReaChES-EYFP and NpHR3.3-p2a-EYFP. bReaChES-EYFP (B) and NpHR3.3-p2a-EYFP (E) had major and minor splice variants from cryptic splicing (noted by #) and exon1 to exon 3 direct splicing (noted by *). C,F) The bReaChES-EYFP intron was moved to an alternative splice site (C). NpHR3.3-p2a-EYFP did not have a separate splice site or degenerate codon options; guided by the published crystal structure, we disrupted the cryptic splice site (F-arrow) by introducing the mutation W179F (F-center), which did not affect opsin function (F-right; p = 0.9754, unpaired t-test). D,G) These iterations of bReaChES-EYFP (D) and NpHR3.3-p2a-EYFP (G) generated either single spliced products (D), or the correct major product and an exon 1-exon 3 minor splice variant (G). H) To catch errors early during scaling/implementation, we have described a protocol for making new INTRSECTs (Fenno et al., 2017) and maintain a Standard Operating Procedure (http://www.optogenetics.org/intrsect_sop.pdf). See Figures s1–s4.
Figure 3.
Figure 3.. Chronic monitoring of viral expression: equivalent INTRSECT and WT expression kinetics.
A) Expression monitoring device: LED light source fed into a filter cube; visible wavelength spectrometer for emission detection. B) Linear input-output relationship between total counted photons (area under the curve - ‘AUC’) of the band-pass filtered signal and integration time set by spectrometer (R2 = 0.9999); spectrometer absorbance (range 0–1) and absolute photons are shown. C-G) Exemplar data: animal co-injected in mPFC with AAV-Con/Fon-EYFP and AAV-Flp-2a-Cre. (C) Wide range of spectrometer integration times ensures a continuous dynamic range (color) of non-zero and non-saturated (grey) signal from early/weak expression through late/strong expression. D) Linear relationship between AUC and integration time in dynamic range of spectrometer maintained in vivo (colors as in C; R2 = 0.9999). E) Expression score: normalizing AUC to integration time and averaging all expression scores for a given time point within spectrometer dynamic range; the time point from panels C and D is noted (arrow). F) Viral expression kinetics model: fit to y = 1−e^(−bx); y: normalized log expression, x: days; b: rate constant (blue dots: within-animal expression scores, red: expression curve fit, dashed: 95% confidence of fit, b = 0.12715, R2 = 0.9474). G) Chronic viral monitoring does not require components beyond typical optogenetic experiments (here, 200um fiber). H) Comparison of WT EYFP expression vs. all three INTRSECT logical expression variants of EYFP co-injected with indicated recombinase viruses. Note high titers of Cre are initially expressed but cause toxicity over time (Con/Foff-EYFP + Cre-green and orange dots), which would not have been apparent without chronic monitoring. Expression kinetics between INTRSECT and non-INTRSECT EYFP viruses are equivalent (comparison of rate constant b between WT and Con/Fon p = 0.4775, WT and Con/Foff p = 0.7728, WT and Coff/Fon p = 0.1380, n = 6 animals per condition, ANOVA with Dunnett’s test). I) Comparison of in vivo expression of all INTRSECT AAV-EYFP variants co-injected with all combinations of AAV recombinases as assayed by confocal fluorescence (6 wks). No difference between expression of WT EYFP and Con/Fon-EYFP (p = 0.7615, unpaired t-test) or WT EYFP and Con/Foff-EYFP (p = 0.2559, unpaired t-test). Coff/Fon-EYFP expression was lower than WT EYFP (EYFP 2.41 × 10e7 a.u. vs. 8.96 × 10e6 a.u., p = 0.0003, unpaired t-test). See also Figures s5 and s6.
Figure 4.
Figure 4.. Identifying and validating a recombinase orthogonal to Cre and Flp.
A) Co-transfected HEK293 cells with combinations of recombinase constructs (rows) and recombinase-dependent EYFP constructs (xDIO-EYFP; columns): flow cytometry. Cre and Dre showed bi-directional cross-activity, and some cross-activity noted when Cre was paired with scDIO-EYFP. VCre showed expected robust action on its vcDIO-EYFP partner without any noted in vitro cross-activity. B) AAV-Cre, -Flp, and -VCre show expected robust activity when co-injected with their partner AAV-xDIO-EYFP without cross-activity after 4 wks in mPFC; low (left) or high (right) magnification. Needle track used to identify injection sites in samples without expression. C) rAAV serotypes of Flp and VCre co-injected with respective AAV-xDIO-EYFP in mPFC, while also injecting AAV-xDIO-EYFP into the VTA. After 2 wks (left), sparse EYFP expression was observed in mPFC and VTA, and at 4 wks high levels of expression in both sites (right).
Figure 5.
Figure 5.. Engineering, optimization, testing, and in vivo function of three-recombinase-dependent Triplesect constructs.
A) Potential intersectional populations available with three-recombinase expression. Cre AND Flp AND VCre intersectional population denoted by central pattern. B) Detailed diagram of EYFP divided into three exons with addition of two introns and recombinase recognition sites (top). The activity of Cre AND Flp AND VCre, reorients exons in the sense direction (middle). Introns are removed during RNA processing (bottom), ending with an intact mRNA encoding EYFP; we labeled this three-recombinase-dependent approach 3x-EYFP. C,D) We generated multiple 3x-EYFP construct variants with different intron placement; variants 1–3 spliced poorly (C), while variant 4 spliced efficiently, as verified by sequencing (bottom); (D) splicing results mirrored by expression patterns in HEK293 cells co-transfected with 3x-EYFP variants and Cre, Flp, and VCre. We therefore used variant 4 going forward. E,F) No expression of 3x-EYFP observed if any of the three recombinases is missing, assayed by flow cytometry of HEK293 cells (E) or in animals injected with 3x-EYFP and recombinases (F; n = 1–2 animals per condition). G-K) 3x engineering approach applied to the calcium sensor GCaMP6m (3x-G6m): similar pattern of proper intron splicing (G) and lack of off-target expression by flow cytometry of HEK293 cells (H; coloring as in E). I) In vitro function of quadruple-transfected (with Cre, Flp, and VCre) neurons expressing 3x-G6m with electrical field stimulation. Population comparison of 3x-G6m vs. WT G6m showed reliable function, albeit with reduced basal fluorescence level (Time-to-peak: 0.3727±0.03938 vs. 0.282±0.01359, p = 0.0045; SNR: 25.01±2.733 vs. 15.72±1.66, p = 0.0080; dF/F: 0.2646±0.03951 vs. 0.0466±0.007635, p < 0.0001; Basal F: 280.6±48.05 vs. 2141±337.2, p < 0.0001; Tau: 1.15±0.1112 vs. 1.736±0.1179, p = 0.0001. 3x-G6m n = 32, WT n = 43, all mean ± S.E.M. Mann-Whitney). J-L) Co-infection of TH-Cre mouse VTA with 3x-G6m and separate viruses encoding Flp and VCre gave rise to robust calcium signals during novel object exploration (J; orange), but not in animals co-infected with 3x-G6m and combinations of only two recombinases did not (green, purple, blue). K) Average Ca2+ signal from traces in J time-locked to onset of novel object exploration (left) show consistent signal from 3x-G6m with all three recombinases, but no signal in two-recombinase controls (total object interactions orange trace = 32, green trace = 27, purple trace = 32, blue trace = 42); (middle) shuffling the bout onset times while maintaining the bout structure in this same trace showed that observed signal was not due to random fluctuation (p <0.01). (right) Average peak signal from traces in K as well as additional three-recombinase-expressing animals (Cre and Flp and VCre active condition vs each two recombinase control p < 0.0001, ANOVA with Dunnett’s test). L) Exemplar slice images (left) and quantification (right) from mice in J: expression of 3x-G6m only (n=3 mice) when all three recombinases are expressed, but not in two-recombinase control mice; Cre and Flp and VCre active condition vs each two-recombinase control p < 0.05, ANOVA with Dunnett’s test; n = 3 sections/mouse, 1 mouse in each of 3 controls, 3 mice in triple-recombinase).
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