All procedures were approved under the University of Colorado’s Institute of Animal Care and Use Committee (IACUC) and performed in the light phase. All authors complied with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.

Prairie voles were bred in-house from colonies originating from Emory University and the University of California Davis, both of which maintain lines descended from wild-caught animals in Illinois (Sue Carter, Courtney Devries and Getz, 1995). These colonies have been used extensively in studies of social behavior and are genetically outbred to preserve natural variation. Voles were weaned at postnatal day 21 and were then housed in standard static rodent cages (17.5L x 9.0w. x 6.0h. in.) in groups of 2–4 with either same sex siblings or same sex voles from similar weaning time frames. Animals were given ad libitum access to water and rabbit chow (5326–3 by PMI Lab Diet). Rabbit chow was supplemented with sunflower seeds, dehydrated fruit bits, and alfalfa cubes. Enriched cages consisted of cotton nestlets, a plastic igloo, and a PVC pipe. Animals were kept in a temperature (23°C–26°C) and humidity-controlled room with a 14:10 h light-dark cycle.

Human embryonic kidney (HEK)293T cells (ATCC, RRID:CVCL_0063) were obtained from the American Type Culture Collection. They were maintained in standard HEK media: DMEM (High Glucose, Pyruvate; Gibco 11995081) supplemented with 10% FBS (Qualified US Origin; BioFluid 200-500-Q) and 1 U/mL Penicillin-Streptomycin (Gibco 15140122). Cells were cultured in T75 or T225 tissue culture flasks and passaged at 70%–80% confluence, with a maximum of 25 passages.

Gene-specific sgRNA targets were designed using Benchling (RRID:SCR_013955), the only platform containing the prairie vole genome (MicOch1.0) for identifying potential off-target effects. sgRNA’s were purchased as oligonucleotides with BbsI cleavage site compatible overhangs from Integrated DNA Technologies (IDT). Oligos comprising sgRNAs were annealed together and cloned into the Bbs1 sites in lenti U6-sgRNA/EF1α-mCherry vector (RRID: Addgene_114199). sgRNA’s for CRISPRa were restricted to −500 bp upstream of the target gene, and sgRNA’s for CRISPRi were restricted to +300 bp downstream of the target gene as per prior recommendations (Maeder et al., 2013; Mali et al., 2013; Konermann et al., 2015). sgRNAs specificity was assessed using National Center for Biotechnology Information’s (NCBI) Basic Local Alignment Search Tool (BLAST). Information related to sgRNA sequences and distance from gene transcription start site (TSS) can be found in Supplementary Table S2. Complete plasmid sequence was confirmed via Plasmidsaurus prior to lentivirus production.

Lentivirus production was performed as specified in Savell et al. (2019a) with some modifications. Briefly, large scale viruses were produced in a sterile environment following Biosafety Level 2 (BSL-2) safety guidelines. HEK-293T cells were transfected with a corresponding CRISPR plasmid: (lenti SYN-FLAG-dCas9-VPR (RRID: Addgene_114196)); lenti SYN-dCas9-KRAB-MeCP2 (RRID: Addgene_155365); lenti U6-sgRNA/EF1a-mCherry (RRID: Addgene_114199)); and psPAX2 packaging plasmid (RRID: Addgene_12260) and the pCMV_VSV-G envelope plasmid (RRID: Addgene_8454) and FuGENE ^® HD Transfection Reagent (Promega) in supplemented Ultraculture media (L-glutamine, sodium pyruvate, and sodium bicarbonate) in a T225 culture flask. Supernatant was passed through a 0.45 µm filter and centrifuged at 106,883 g (38,100 rpm) for 65 min at 4°C in a 70Ti fixed angle titanium rotor (Beckmann Coulter). The viral pellet was resuspended in 1/100th supernatant volume of sterile PBS and stored at −80°C. Physical viral titer was determined using Lenti-X qRT-PCR Titration kit (Takara) and only viruses with >1 × 10⁹ GC/mL were used. Viruses were stored in sterile aliquots of PBS at −80°C.

Sexually naïve adult prairie voles were anesthetized with isoflurane (4% induction and 1.5%–2.5% maintenance) at an oxygen flow rate of 1L/min and secured in a head-fixed stereotaxic frame (Kopf Instruments). Female reproductively intact animals were used for all experiments, except those targeting Avpr1a, for which males were used. In total, 65 animals were used across all experimental groups. Body temperature was maintained at 37°C using a closed loop heating pad with a rectal thermometer. Eyes were lubricated with ophthalmic ointment (Sterile Lubricant Eye Ointment), and depth of anesthesia was monitored by breathing and toe and tail pinch response. Using a shaver, fur was removed from the dorsal portion of the head and, a midline incision was made and disinfected with 70% isopropyl alcohol followed by 10% betadine. Briefly, the scalp and connective tissue were removed above the frontal skull plates and the head leveled in the anterior-posterior plane. Guide holes were drilled using stereotaxic coordinates (all coordinates in respect to bregma (Paxinos and Watson, 2007): nucleus accumbens core and shell: AP: +1.7; ML ±: 1.0; ventral pallidum: AP: +1.5; ML ±: 0.9; All infusions were made under anesthesia using a gastight 30-gauge stainless steel injection needle (Hamilton Syringes) that extended into the infusion site. Bilateral lentivirus microinfusions were made using a UMP3-T syringe pump (World Precision Instruments) at a rate of 2 nL/s at DV: −4.8/4.7/4.6 mm for the nucleus accumbens or DV: −5.8/5.7/5.6 mm for the ventral pallidum. At each DV coordinate, 700/700/600 nL were injected for a total of 2 uL per hemisphere (See Supplementary Figure S1 for virus volume validation). Injection needles remained in place for 10 min post infusion to allow time for diffusion. Prairie voles were infused bilaterally with 2 uL of total lentivirus mix which comprised of a 1:10 ratio of sgRNA virus to dCas9-VPR or KRAB-MeCP2 virus in sterile PBS. Surgical incisions sites were closed with vicryl sutures. Animals received extended release meloxicam (4 mg/kg), lidocaine at the incision site, and saline (1 mL) for pain, and post-operative management. Animals recovered 72 h in a BSL-2 facility to minimize any exposure to animal shedding of lentivirus particles.

For samples used in qRT-PCR, animals were euthanized via rapid decapitation. Brains were immediately extracted and rinsed in sterile saline. Fluorescent guided dissections were performed manually using sterile RNase free razor blades and mCherry fluorescence was visualized with a fluorescent dissecting microscope Olympus MVX10 MacroZoom at the University of Colorado Boulder MCDB Light Microscopy Core Facility (RRID:SCR_018993). During dissection, tissue was kept on ice blocks and in cold saline. For each animal, the right and left hemispheres received different viral constructs (targeting sgRNA vs. LacZ control), and each hemisphere was dissected separately. No pooling was performed. Each data point in the qRT-PCR dataset represents tissue from a single hemisphere of an individual animal. Because viral constructs were injected into opposite hemispheres, each animal served as its own control, minimizing variability due to individual differences in gene expression or viral transduction. Dissected hemispheres were flash frozen on dry ice and stored at −80°C until RNA extraction.

For samples used for immunohistochemistry, animals were injected with 0.15–0.30 mL 1:2 ketamine/xylazine and transcardially perfused with 4% paraformaldehyde (PFA, Electron Microscopy Scienes) in phosphate buffered saline. Brains were kept in 4% PFA overnight and transferred to 30% sucrose. After brains sank, they were rinsed and flash frozen on dry ice and stored at −80°C for 24 h prior to sectioning on a freezing microtome (Leica June SM2000R, 40 µm/slice).

Samples were processed for total purified RNA as described in Cunningham et al. (2019) using the Norgen Total RNA/gDNA kit (Norgen Biotek no. 48700). Briefly, frozen tissue was placed in 600 µL ice cold lysis buffer and homogenized using a Scilogex homogenizer. Homogenized tissue was kept on wet ice while all animals for the cohort were processed. The homogenizer tip was thoroughly cleaned in between each animal with DEPC-treated water and 70% ethanol to prevent cross contamination across samples. Homogenized samples were centrifuged at 4000 g for 10 min and supernatant was transferred to new pre-chilled tubes.

Total RNA (2 uL for each sample) was Nanodropped to determine RNA concentration (Agilent Technologies). RNA samples were all diluted to 25 ng/μL with RNAse-free water (Norgen). cDNA was generated using high Capacity cDNA Reverse Transcription kit (Applied Bio Systems). For each sample, cDNA was produced using a mixture of 2 μL of 10X RT buffer, 0.8 μL of dNTP, 2 μL of random primers, 1 μL of Transcriptase, 1 μL of RNA inhibitor, and 3.2 μL of ddH2O. RNA (250 ng) was then added, and cDNA was generated using reaction conditions 25°C for 10 min, 2 × (37°C for 120 min, 85°C for 5 min). Samples were stored at −20°C until processing.

Prairie vole gene expression for Oxtr, Avpr1a, Drd1, Drd2, and Gapdh was quantified using qPCR. Primer sequences are provided in Supplementary Table S3. Samples and probes were processed in triplicate in MicroAmp Fast Optical 96 well Reaction Plate (Applied Bio Systems) with 0.66 µL of cDNA, 0.50 µL of probe, 5 µL of TaqMan Fast Advanced Master Mix (Applied Biosystems), and 3.84 µL of ddH₂O per well for a total volume of 10 µL. Plates were covered with Optical Adhesive Film (Applied Biosystems), vortexed, and centrifuged prior to PCR amplification in Applied Biosystems QuantSTudio 3 qPCR machine (Applied Biosystems). DNA was amplified using the following cycling conditions: 50°C for 2 min, 95°C for 20 s, and 40 cycles of 95°C for 1 s and 60°C for 20 s.

The mean cycle threshold (Ct) value was calculated for each sample run in triplicate. Triplicates with a standard deviation greater than 0.30 were assessed for potential outliers. In cases where a clear outlier could be visually identified (e.g., one Ct value substantially deviating from the other two), that value was excluded and the remaining two values were averaged. If no clear outlier was apparent, the entire sample was excluded to avoid introducing bias. In total, 44 out of 864 wells (5.1%) were excluded from analysis based on this criterion, spanning multiple PCR plates and experiments. The exclusion rate is within the expected range for qPCR experiments and reflects a conservative quality control approach to ensure the accuracy and reliability of reported expression values. Gene expression quantification was performed using the ΔΔCt method (Livak and Schmittgen, 2001), with all samples normalized to Gapdh.

Immunohistochemistry was performed to visualize the FLAG tag on dCas9-VPR and dCas9-KRAB-MeCP2 constructs. Free-floating brain slices from PFA-perfused prairie voles were stored at −20°C in cryoprotectant consisting of 50% glycerol in 0.05 M phosphate buffer. For immunofluorescent labeling of FLAG, sections were washed in 1X PBS, for 30 min, with fresh PBS every 5 min. Sections were blocked with 5% normal donkey serum (Jackson Laboratories) containing 0.2% Triton X-100 for 1 h followed by primary antibody incubation with mouse-anti-FLAG (Invitrogen, RRID:AB_1957945, 1:100) at 4°C for 40 h in 750 uL of solution. After washing, sections were incubated with AlexaFluor 488 secondary antibody (Life Technologies, 1:500) for 1 h. Slices underwent another series of washes and were then mounted on slides and coverslipped with Prolong Gold Antifade Mountant (Life Technologies) to preserve signal intensity and brightness.

Slices were imaged using a Nikon A1 Laser Scanning confocal Microscope at the University of Colorado Boulder MCDB Light Microscopy Core Facility (RRID:SCR_018993). Confocal images were taken with a 20x lens in two channels (green and red) of the nucleus accumbens and ventral pallidum. Confocal stacks were projected as single images using maximum fluorescence.

Data are shown as means ± standard error of the mean. Statistical significance α was set at 0.05. All n values represent number of animals. Statistical analyses were carried out using either GraphPad PRISM (version 10.4.1, Graphpad, San Diego, CA) or Python (v 3.12.4) in a reproducible computing environment. Python scripts were executed in Juypter Notebook (v 7.0.8) via Anaconda Navigator (v 2.6.4). Paired t-tests with error propagation were performed.

We employed a dual-vector system to enable gene activation or repression. One lentiviral construct encoded either dCas9-VPR (Figure 1A) or dCas9-KRAB-MeCP2 (Figure 1B) under the control of the neuron-specific synapsin (SYN) promoter. The second construct carried a U6-driven sgRNA targeting either Oxtr, Avpr1a, Drd1, Drd2, or a non-targeting lacZ control, and included a mCherry reporter driven by the EF1α promoter for visualization of sgRNA-expressing cells (Figures 1A,B). Following lentiviral injection, animals were given 21 days to allow for both recovery and robust expression of CRISPRa/i constructs (Figure 1C), after which immunohistochemistry (IHC) was performed to confirm expression in targeted brain regions, the nucleus accumbens (Figure 1D) or ventral pallidum (Figure 1E).

Representative images demonstrate detection of constructs in the nucleus accumbens (Figure 1F) and the ventral pallidum (Figure 1G). Specifically, FLAG-tagged dCas9-VPR (CRISPRa) and FLAG-tagged dCas9-KM (CRISPRi) were detected in transduced neurons (Figures 1F,G, left panels, respectively). Co-expression of U6-driven sgRNA’s, co-labeled with a mCherry reporter under an EF1α, promoter was observed in the same regions (Figures 1F,G, middle panels). Merged images confirm colocalization of dCas9 constructs and sgRNA’s indicating successful neuronal expression of both components (Figures 1F,G, right panels).

These findings validate expression of lentiviral CRISPRa/i in a non-traditional mammalian model, demonstrating effective lentiviral-mediated delivery and neuronal expression of CRISPRa and CRISPRi constructs in the prairie vole brain across two brain regions.

To evaluate the effectiveness of CRISPRa and CRISPRi in the prairie vole brain, we focused on four target genes implicated in social behavior: the nonapeptide receptors Oxtr and Avpr1a, and the two most abundant dopamine receptors, Drd1 and Drd2. We targeted these genes in mesolimbic brain regions where they are highly expressed and known to modulate pair bonding behaviors (Wang et al., 1999; Young, 1999; Gingrich et al., 2000; Young et al., 2001; Aragona et al., 2006; Gobrogge and Wang, 2016; Loth and Donaldson, 2021; Pierce et al., 2024). Specifically, Oxtr, Drd1, and Drd2 were targeted in the nucleus accumbens (NAc), while Avpr1a was targeted in the ventral pallidum (VP).