DOI: 10.5061/dryad.9zw3r22jn
title: Data for: An AAV-CRISPR/Cas9-strategy for gene editing across divergent rodent species: Targeting neural oxytocin receptors as a proof of concept

description: 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 &gt;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.
description: 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 (<em>39</em>). 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 I<sup>125</sup>-OVTA
 (2200 Ci/mmol, ornithine vasotocin analog, #NEX254010UC, PerkinElmer, MA,
 USA) or 50 pM I<sup>125</sup>-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%
 MgCl<sub>2</sub> 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. I<sup>125</sup>-activity (dpm) was
 calculated using an I<sup>125-</sup>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
 <em>blastn</em> 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.
description: Tables (.csv) can be opened with any standard software, such as
 excel. Code is written for R/R-studo