A major issue in neuroscience is the poor translatability of research results from preclinical studies in animals to clinical outcomes. Comparative neuroscience can overcome this barrier by studying multiple species to differentiate between species-specific and general mechanisms of neural circuit functioning. Targeted manipulation of neural circuits often depends on genetic dissection, and use of this technique has been restricted to only a few model species, limiting its application in comparative research. However, ongoing advances in genomics make genetic dissection attainable in a growing number of species. To demonstrate the potential of comparative gene editing approaches, we developed a viral-mediated CRISPR/Cas9 strategy that is predicted to target the oxytocin receptor (Oxtr) gene in >80 rodent species. This strategy specifically reduced OXTR levels in all evaluated species (n=6), without causing gross neuronal toxicity. Thus, we show that CRISPR/Cas9-based tools can function in multiple species simultaneously. Thereby, we hope to encourage comparative gene editing and improve the translatability of neuroscientific research.

OXTR and AVPR1A autoradiography

Fresh frozen brains were sectioned on a cryostat (Epredia Cryostar NX-70, Thermo-Scientific, MA, USA) at 20 μm, mounted on Superfrost Plus slides (Fisher Scientific) and stored at -80C until use. Autoradiography was performed as previously described (39). Briefly, slides were thawed and fixed for two minutes in 0.1% paraformaldehyde in phospate-buffered saline (PBS) for two minutes, washed in 50 mM Tris in PBS (pH 7.4, 2x10 minutes) and incubated in 50 mM Tris buffer, supplemented with 0.1% BSA and 50 pM I¹²⁵-OVTA (2200 Ci/mmol, ornithine vasotocin analog, #NEX254010UC, PerkinElmer, MA, USA) or 50 pM I¹²⁵-AVP (2200 Ci/mmol, linear arginine vasopressin, #NEX310010UC, PerkinElmer) at room temperature for one hour. Unbound ligand was removed by washing in 50 mM Tris with 0.2% MgCl₂ at 4 degrees (4x5 minutes) and 30 minutes at RT. Slides were dipped in miliQ, dried, and placed in a cassette with BioMax MR film (Sigma-Aldrich, MO, USA). After 7 days, films were developed and imaged using an MCID core system (Interfocus Co., UK).   Mean gray values of viral-targeted regions, corrected for background, were determined in ImageJ. I¹²⁵-activity (dpm) was calculated using an I^125-standard and taken as a proxy for OXTR or AVPR1A density. Differences in OXTR or AVPR1A density were determined by comparing protein density levels in AAV-ΔOXTR injected regions to protein density levels in contralateral AAV-CTRL injected regions.

Immunohistochemistry

Animals were deeply anesthetized and transcardially perfused with PBS, followed by PBS supplemented with 4% paraformaldehyde. Brains were sectioned on a cryostat (Cryostar NX-70) at 40 um and stored in cryoprotectant buffer at -20C until use. Sections were thawed, washed in PBS, and blocked and permeabilized in PBS supplemented with 0.1% tween (PBST) and 5% normal donkey serum for 1 hour at RT. Next, sections were incubated in PBST supplemented with 0.5% rabbit anti-NeuN (1:1000, AB104225, Abcam, UK) at 4C overnight. After PBS washes, sections were incubated in anti-rabbit Alexa Fluor 568 antibodies (1:500, Molecular Probes, OR. USA) for 1h. Sections were mounted and coverslipped in Fluoromount-G containing DAPI (Thermo-Scientific). Z-stacks with 60X magnification of eGFP-injected regions and the corresponding sham-injected contralateral region were imaged using a Keyence microscope. Z-stack were projected with maximal intensity and NeuN-positive cells were manually counted in ImageJ by a blinded observer.       

BLAST

We used the blastn algorithm in the NCBI BLAST+ software suite to search for perfect alignment of the target RNA sequences plus the permissive PAM-sequence (5-NGG-3”) to rodent (taxid 9989) and mammalian (taxid 40674) genomes to identify species in which our tool is predicted to be functional.

Tables (.csv) can be opened with any standard software, such as excel. Code is written for R/R-studo