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Optogenetics rAAVs

INTRSECT (INTronic Recombinase Sites Enabling Combinatorial Targeting)-related constructs

A versatile single-AAV system for selective expression that is conditional upon multiple cell-type features (such as wiring or genetic type) related by Boolean logic operations (AND, AND NOT) using multiple recombinases including Cre and Flp. May be used in combination with multiple viruses, transgenic animals, or combinations thereof. The engineered INTRSECT introns that enable this targeting can also be applied to other genes in a variety of settings, and the introns themselves enhance expression of the host gene.

pAAV-Ef1a-Cre-WPRE [ Vector Map ]
pAAV-Ef1a-Dre-WPRE [ Vector Map ]
pAAV-nEF-Con/Fon hChR2(H134R)-EYFP-WPRE [ Vector Map ]
pAAV-nEF-Con/Foff hChR2(H134R)-EYFP-WPRE [ Vector Map ]
pAAV-nEF-Coff/Fon hChR2(H134R)-EYFP-WPRE [ Vector Map ]
pAAV-hSyn-Con/Fon EYFP-WPRE [ Vector Map ]
pAAV-hSyn-Con/Fon hChR2(H134R)-EYFP-WPRE [ Vector Map ]
pAAV-hSyn-Con/Foff hChR2(H134R)-EYFP-WPRE [ Vector Map ]
pAAV-hSyn-Con/Foff EYFP-WPRE [ Vector Map ]
pAAV-hSyn-Coff/Fon EYFP-WPRE [ Vector Map ]
pAAV-hSyn-Coff/Fon hChR2(H134R)-EYFP-WPRE [ Vector Map ]
pAAV-Ef1a-vCre-WPRE [ Vector Map ]
pAAV-EF1a-vCreDIO hChR2(H134R)-EYFP-WPRE [ Vector Map ]
pAAV-Ef1a-sCre-WPRE [ Vector Map ]
pAAV-EF1a-sCreDIO hChR2(H134R)-EYFP-WPRE [ Vector Map ]
pAAV-EF1a-mCherry-IRES-Flpo-WPRE [ Vector Map ]
pAAV-EF1a-mCherry-IRES-Cre-WPRE [ Vector Map ]
pAAV-EF1a-mCherry-IRES-Dre-WPRE [ Vector Map ]
pAAV-EF1a-mCherry-IRES-sCre-WPRE [ Vector Map ]
pAAV-EF1a-mCherry-IRES-vCre-WPRE [ Vector Map ]
pAAV-Ef1a-Flpo-WPRE [ Vector Map ]
pAAV-Ef1a-fDIO-EYFP-WPRE [ Vector Map ]
pAAV-EF1a-fDIO-hChR2(H134R)-EYFP-WPRE [ Vector Map ]
pAAV-Ef1a-dDIO hChR2(H134R)-EYFP-WPRE [ Vector Map ]

SwiChR and iC1C2: Action potential inhibition with chloride-conducting channelrhodopsins

The engineered iC1C2 was designed based on the 2012 crystal structure of C1C2 to conduct chloride ions instead of cations, utilizing physiological chloride gradients to precisely inhibit action potentials in response to blue light. The resulting inhibition is much more light-sensitive than with prior optogenetic inhibitory tools and involves reversible input resistance changes [ Berndt et al. 2014 ]. Neuronal inhibition can also be controlled by a switchable variant: Step-Waveform Inhibitory ChannelRhodopsin (SwiChR), which is activated by brief blue light stimulation at low intensities, remains open in the dark for an extended period of time and gets deactivated by red light. Light sensitivity of expressing cells is further improved. The channel pore is open and flow of chloride ions across the cell membrane is elevated between the blue and red light pulses, thereby greatly reducing spike probability in expressing neurons without the need for continuous light delivery.

pAAV-CaMKIIa-iC1C2 2.0-TS-EYFP [ Vector Map ]
pAAV-CaMKIIa-SwiChRCA-TS-EYFP [ Vector Map ]
pAAV-EF1a-DIO iC1C2 2.0-TS-eYFP-WPRE [ Vector Map ]
pAAV-EF1a-DIO SwiChRCA-TS-eYFP-WPRE [ Vector Map ]

Cell-filling variants

Cell filling variants of optogenetic tools use p2A sequences for bicistronic expression of opsin and fluorophore from a single virus to allow for enhanced identification of opsin-expressing cells without a loss in functional opsin expression. All of the constructs have been deposited at the UNC and Stanford Vector Cores. Please check their websites for availability of prepackaged AAV-2, AAV-5 or AAV-DJ

pAAV-CamKII-hChR2 (T159C)-p2A-EYFP-WPRE [ Vector Map ] [UNC/Stanford]
pAAV-CamKII-hChR2 (T159C)-p2A-mCherry-WPRE [ Vector Map ] [UNC/Stanford]
pAAV-CamKII-hChR2 (E123T/T159C)-p2A-EYFP-WPRE [ Vector Map ] [UNC/Stanford]
pAAV-CamKII-hChR2 (E123T/T159C)-p2A-mCherry-WPRE [ Vector Map ] [UNC/Stanford]
pAAV-CamKIIa-C1V1 (E162T)-TS-p2A-EYFP-WPRE [ Vector Map ] [UNC/Stanford]
pAAV-CamKIIa-C1V1 (E162T)-TS-p2A-mCherry-WPRE [ Vector Map ] [UNC/Stanford]
pAAV-CamKIIa-C1V1 (t/t)-TS-p2A-EYFP-WPRE [ Vector Map ] [UNC/Stanford]
pAAV-CamKIIa-C1V1 (t/t)-TS-p2A-mCherry-WPRE [ Vector Map ] [UNC/Stanford]
pAAV-Ef1a-DIO hChR2 (E123T/T159C)-p2A-EYFP-WPRE [ Vector Map ] [UNC/Stanford]
pAAV-Ef1a-DIO hChR2 (E123T/T159C)-p2A-mCherry-WPRE [ Vector Map ] [UNC/Stanford]
pAAV-Ef1a-DIO C1V1 (E162T)-TS-p2A-EYFP-WPRE [ Vector Map ] [UNC/Stanford]
pAAV-Ef1a-DIO C1V1 (E162T)-TS-p2A-mCherry-WPRE [ Vector Map ] [UNC/Stanford]
pAAV-Ef1a-DIO C1V1 (t/t)-TS-p2A-EYFP-WPRE [ Vector Map ] [UNC/Stanford]
pAAV-Ef1a-DIO C1V1 (t/t)-TS-p2A-mCherry-WPRE [ Vector Map ] [UNC/Stanford]
pAAV-CamKIIa-hChR2(C128A/H134R)-p2A-EYFP [ Vector Map ] [UNC/Stanford]
pAAV-Ef1a-DIO hChR2(C128A/H134R)-p2A-EYFP [ Vector Map ] [UNC/Stanford]

Red-Shifted Optical Excitation: C1V1 variants C1V1

Combinatorial optogenetic excitation within intact mammalian tissues: a new family of engineered chimeric opsin variants (C1V1) composed of ChR1 and VChR1 fragments, that implements fast and potent optical excitation at red-shifted wavelengths.

pLenti-CaMKIIa-C1V1-EYFP [ Vector Map ]
pLenti-CaMKIIa-C1V1-TS-EYFP [ Vector Map ]
pLenti-CaMKIIa-C1V1 (E122T)-TS-EYFP [ Vector Map ]
pLenti-CaMKIIa-C1V1 (E162T)-TS-EYFP [ Vector Map ]
pLenti-CaMKIIa-C1V1 (E122T/E162T)-TS-EYFP [ Vector Map ]
pAAV-CaMKIIa-C1V1 (E122T/E162T)-TS-EYFP [ Vector Map ] [UNC]
pAAV-CaMKIIa-C1V1 (E122T/E162T)-TS-mCherry [ Vector Map ] [UNC]
pAAV-Ef1a-DIO-C1V1 (E122T/E162T)-TS-EYFP [ Vector Map ] [UNC]
pAAV-Ef1a-DIO C1V1 (E122T/E162T)-TS-mCherry [ Vector Map ] [UNC]
pAAV-CaMKIIa-C1V1(E162T)-p2A-EYFP [ Vector Map ]

Stabilized Step Function Opsins SSFO

ChR2 variant harboring two amino acid substitutions which act to stabilize the conducting state of the channel to deactivate with a time constant of nearly 30 minutes following a brief pulse of activating blue light. Like previously published step function opsins, this stabilized step function opsin (SSFO) may be deactivated using yellow light (590nm). The stabilized open state of the channel allows for both lower power activation, meaning in some circumstances the light delivery system need not penetrate the brain, as well as for behavior in the absence of a tethered laser or other light delivery system.

pLenti-CaMKIIa-hChR2 (C128S/D156A)-EYFP [ Vector Map ]
pAAV-EfIa-SSFO-EYFP [ Vector Map ] [UNC]
pAAV-hSyn-SSFO-EYFP [ Vector Map ] [UNC]
pAAV-CaMKIIa-hChR2 (C128S/D156A)-EYFP [ Vector Map ] [UNC]
pAAV-CaMKIIa- hChR2 (C128S/D156A)-mCherry [ Vector Map ] [UNC]
pAAV-Ef1a-DIO hChR2 (C128S/D156A)-EYFP [ Vector Map ] [UNC]
pAAV-Ef1a-DIO hChR2 (C128S/D156A)-mCherry [ Vector Map ] [UNC]

Second-generation Ultrafast Optogenetic Control

pLenti-CaMKIIa-hChR2(T159C)-EYFP-WPRE [ Vector Map ]
pLenti-CaMKIIa-hChR2(E123T/T159C)-EYFP-WPRE [ Vector Map ]
pAAV-CaMKIIa-hChR2 (E123T/T159C)-EYFP [ Vector Map ] [UNC]
pAAV-CaMKIIa-hChR2 (E123T/T159C)-mCherry [ Vector Map ] [UNC]
pAAV-CaMKII-hChR2 (T159C)-EYFP-WPRE [ Vector Map ] [UNC]
pAAV-CaMKII-hChR2 (T159C)-mCherry-WPRE [ Vector Map ] [UNC]
pAAV-CaMKII-hChR2 (E123A)-EYFP-WPRE [ Vector Map ] [UNC]
pAAV-CaMKII-hChR2 (E123A)-mCherry-WPRE [ Vector Map ] [UNC]
pAAV-EF1a-DIO-hChR2 (E123T/T159C)-EYFP [ Vector Map ] [UNC]
pAAV-EF1a-DIO-hChR2 (E123T/T159C)-mCherry [ Vector Map ] [UNC]
pAAV-EF1a-DIO-ChR2 (T159C)-mCherry-WPRE [ Vector Map ] [UNC]
pAAV-EF1a-DIO-ChR2 (T159C)-EYFP-WPRE [ Vector Map ] [UNC]
pAAV-EF1a-DIO-ChR2 (E123A)-EYFP-WPRE [ Vector Map ] [UNC]
pAAV-EF1a-DIO-ChR2 (E123A)-mCherry-WPRE [ Vector Map ] [UNC]

Transsynaptic Tracers: WGA-Cre

Transsynaptic tracing viruses are available to assist in targeting neuronal subpopulations based on their syanptic connectivity to a downstream region. These may be used in conjunction with any of the DIO class of targeting virus. Researchers need to determine the directionality and extent of spread of the WGA-cre translocation in each experimental system.

pAAV-EF1a-mCherry-IRES-WGA-Cre [ Vector Map ] [UNC]

Third-generation Optogenetic Inhibition: eNpHR 3.0

Engineered halorhodopsin construct for nanoamp-scale optical inhibition with chloride currents at low light powers (<5 mW/mm2) suitable for in vivo use. Reversible, step-like kinetic stability over many minutes suitable for physiology or behavior, responsive to far-red light, and well-tolerated due to enhanced membrane trafficking modifications.

pAAV-Ef1a-DIO-eNpHR 3.0-EYFP [ Vector Map ] [UNC]
pAAV-Ef1a-DIO-eNpHR 3.0-mCherry [ Vector Map ] [UNC]
pAAV-CaMKIIa-eNpHR3.0-EYFP [ Vector Map ] [UNC]
pAAV-CaMKIIa-eNpHR3.0-mCherry [ Vector Map ] [UNC]
pAAV-hSyn-eNpHR 3.0-EYFP [ Vector Map ] [UNC]
pAAV-hSyn-eNpHR 3.0-mCherry [ Vector Map ] [UNC]
pAAV-EF1a-eNpHR 3.0-EYFP [ Vector Map ]
pAAV-hThy1-eNpHR 3.0-EYFP [ Vector Map ] [UNC]
pLenti-CaMKIIa-eNpHR 3.0-EYFP [ Vector Map ]
pLenti-hSyn-eNpHR3.0-EYFP [ Vector Map ]

Third-generation Optogenetic Inhibition: Arch 3.0 ArchT 3.0, and Mac 3.0

Optical inhibition with enhanced proton pumps, Arch from H. sodomense, ArchT from Halorubrum sp. TP009 and Mac from L. maculans modified for mammalian expression with ER export and Trafficking Signals resulting in 3-5 fold increase in originally reported currents and stable in vivo expression. [Technical Paper]

pAAV-Ef1a-DIO eArch 3.0-EYFP [ Vector Map ]
pAAV-CaMKIIa-eArchT 3.0-EYFP [ Vector Map ]
pLenti-CaMKIIa-eArch 3.0-EYFP [ Vector Map ]
pLenti-CaMKIIa-eArchT 3.0-EYFP [ Vector Map ]
pLenti-CaMKIIa-eMac 3.0-EYFP [ Vector Map ]
pAAV-CaMKIIa-eArch 3.0-EYFP [ Vector Map ] [UNC]
pAAV-hThy1-eArch 3.0-EYFP [ Vector Map ]
pAAV-hSyn-eArch 3.0-EYFP [ Vector Map ]

Ultrafast Optogenetic Control: ChETA

Engineered channelrhodopsin-2 variant with faster deactivation kinetics, resulting in 1) high-fidelity light-driven spiking over sustained trains at least up to 200 Hz, 2) reduced multiplets and plateau potentials, 3) faster recovery from inactivation, and 4) improved temporal stationarity of performance in sustained trains.

pLenti-CaMKIIa-hChR2(E123T-H134R)-EYFP [ Vector Map ]
pAAV-Ef1a-DIO-ChETA-EYFP [ Vector Map ] [UNC]

Optical Control of Intracellular Signaling: Opto-XRs

Chimeric fusions of bovine Rhodopsin and adrenergic G-Protein Coupled Receptors allowing optical control of GPCR signaling cascades. Proteins are activated by 500nm light.

pcDNA3.1v5his-opto-a1AR-EYFP [ Vector Map ]
pcDNA3.1v5his-opto-b2AR-EYFP [ Vector Map ]
pAAV-CaMKIIa-Opto A1-EYFP [ Vector Map ]
pAAV-CaMKIIa-Opto B2-EYFP [ Vector Map ]
pAAV-CaMKIIa-Opto D1-EYFP [ Vector Map ]
pAAV-EF1a-DIO Opto A1-EYFP [ Vector Map ]
pAAV-EF1a-DIO Opto A1-mCherry [ Vector Map ]
pAAV-EF1a-DIO Opto B2-EYFP [ Vector Map ]
pAAV-EF1a-DIO Opto B2-mCherry [ Vector Map ]
pAAV-EF1a-DIO Opto D1-EYFP [ Vector Map ]
pAAV-EF1a-DIO Opto D1-mCherry [ Vector Map ]
pAAV-hSyn Opto A1-EYFP [ Vector Map ]
pAAV-hSyn Opto B2-EYFP [ Vector Map ]
pAAV-hSyn Opto D1-EYFP [ Vector Map ]

Bi-stable excitation: Step Function Opsins (SFOs)

Three point-mutants of humanized ChR2 convert a brief pulse of light into a stable step in membrane potential. The lentiviral vectors were created by site-directed mutagenesis of the C128 position in ChR2. All three mutants are activated by blue (470nm) light. Photocurrents generated by ChR2(C128A) and ChR2(C128S) can be effectively terminated by a pulse of green (542nm) light.

pLenti-CaMKIIa-hChR2(C128A)-EYFP-WPRE [ Vector Map ]
pLenti-CaMKIIa-hChR2(C128S)-EYFP-WPRE [ Vector Map ]
pLenti-CaMKIIa-hChR2(C128T)-EYFP-WPRE [ Vector Map ]

Cre-inducible Adeno-associated Virus: DIO-AAV

For cell type-specific targeting and to capitalize on the large number of Cre driver lines with the flexible virus injection/fiberoptic approach, we have developed a tool we call DIO-AAV (doublefloxed inverse orf) AAV.

pAAV-Ef1a-DIO-hChR2(H134R)-EYFP-WPRE-pA [ Vector Map ] [UNC]
pAAV-Ef1a-DIO-hChR2(H134R)-mCherry-WPRE-pA [ Vector Map ] [UNC]
pAAV-Ef1a-DIO-eNpHR-EYFP-WPRE-pA [ Vector Map ]

Optical Excitation: Channelrhodopsin-2 (ChR2)

There are two versions of the ChR2 sequence, one containing the wildtype sequence and another containing condons optimized for mammalian expression (hChR2). In-frame fusions to mCherry or EYFP are available to make visualization of ChR2-expressing cells easier. ChR2 and XFP are fused via a NotI site. The linker is GCGGCCGCC.

ChR2-XFPs are available either in a standard mammalian expression vector containing the CMV promoter or in lentiviral expression vectors under the control of the ubiquitous EF-1a or the neuron-specific CaMKIIa or human Synapsin I promoters. The structure of the lentivirus is shown below.

pcDNA3.1/hChR2-mCherry [ Vector Map ]
pcDNA3.1/hChR2-EYFP [ Vector Map ]
pcDNA3.1/hChR2(H134R)-EYFP [ Vector Map ]
pAAV-CaMKIIa-hChR2(H134R)-EYFP [ Vector Map ] [UNC]
pAAV-CaMKIIa-hChR2(H134R)-mCherry [ Vector Map ] [UNC]
pAAV-hSyn-hChR2(H134R)-EYFP [ Vector Map ] [UNC]
pAAV-hSyn-hChR2(H134R)-mCherry [ Vector Map ] [UNC]
pAAV-hSyn-hChR2(H134R)-WPRE [ Vector Map ]
pAAV-hSyn-hChR2(H134R)-p2A-WPRE [ Vector Map ]
pAAV-mThy1-hChr2(H134R)-eYFP-WPRE [ Vector Map ]
pAAV-hThy1-hChR2(H134R)-eYFP-WPRE [ Vector Map ]
pAAV-GFAP-hChR2(H134R)-EYFP [ Vector Map ]
pAAV-GFAP-hChR2(H134R)-mCherry [ Vector Map ]
pAAV-EF1a-hChR2(H134R)-EYFP [ Vector Map ]
pLenti-EF1a-hChR2-EYFP-WPRE (a.k.a. pLECYT) [ Vector Map ]
pLenti-EF1a-hChR2(H134R)-EYFP-WPRE (a.k.a. pLECYT) [ Vector Map ]
pLenti-CaMKIIa-hChR2-mCherry-WPRE [ Vector Map ]
pLenti-CaMKIIa-hChR2-EYFP-WPRE [ Vector Map ]
pLenti-Hcrt-hChR2(H134R)-EYFP [ Vector Map ]
pLenti-Synapsin-hChR2(H134R)-EYFP-WPRE [ Vector Map ]
pLenti-mThy1-hChR2(H134R)-eYFP [ Vector Map ]
pLenti-hThy1-hChR2(H134R)-eYFP [ Vector Map ]

Optical Inhibition: Halorhodopsin (NpHR)

The NpHR sequence here has been optimized for mammalian expression. The NpHR-EYFP inframe fusion genes are made via a NotI site with the linker GCGGCCGCC. The start codon on EYFP has been deliberately removed. To reduce membrane blebbing or other toxicity at high levels of expression, we have generated a modified eNpHR by adding signaling peptides to enhance membrane translocation and ER export.

Currently, the only versions of halorhodopsin that are available for shipping are eNpHR3.0 (above) and eNpHR2.0 (pLenti-CaMKII-eNpHR-EYFP). eNpHR2.0 may be superior in some cell types, including photoreceptors (Busskamp et al., Science 2010).

pLenti-CaMKIIa-eNpHR2.0-EYFP-WPRE [ Vector Map ]
pAAV-mThy1-eNpHR 2.0-eYFP-WPRE [ Vector Map ]
pAAV-hThy1-eNpHR 2.0-eYFP-WPRE [ Vector Map ]
pAAV-hSyn-NpHR-WPRE [ Vector Map ]
pLenti-hThy1-eNpHR 2.0-eYFP [ Vector Map ]

Optical Excitation: Volvox Channelrhodopsin-1 (VChR1)

The VChR1 sequence here has been optimized for mammalian expression. The VChR1-EYFP inframe fusion genes are made via a NotI site with the linker GCGGCCGCC. The start codon on EYFP has been deliberately removed. VChR1-EYFP are also available in a standard mammalian expression vector and lentivirus vectors.

pcDNA3.1/VChR1-EYFP [ Vector Map ]
pcDNA3.1/VChR1-mCherry [ Vector Map ]
pLenti-CaMKIIa-VChR1-EYFP-WPRE [ Vector Map ]
pLenti-CaMKIIa-VChR1-mCherry-WPRE [ Vector Map ]

Control Fluorophores

pAAV-CaMKIIa-EYFP [ Vector Map ] [UNC]
pAAV-CaMKIIa-mCherry [ Vector Map ] [UNC]
pAAV-Ef1a-DIO-EYFP-WPRE-pA [ Vector Map ] [UNC]
pAAV-Ef1a-DIO mCherry [ Vector Map ] [UNC]
pAAV-hSyn-EYFP [ Vector Map ]

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