Oxytocin receptor knockout prairie voles generated by CRISPR/Cas9 editing show reduced preference for social novelty and exaggerated repetitive behaviors

Abstract

Behavioral neuroendocrinology has benefited tremendously from the use of a wide range of model organisms that are ideally suited for particular questions. However, in recent years the ability to manipulate the genomes of laboratory strains of mice has led to rapid advances in our understanding of the role of specific genes, circuits and neural populations in regulating behavior. While genome manipulation in mice has been a boon for behavioral neuroscience, the intensive focus on the mouse restricts the diversity in behavioral questions that can be investigated using state-of-the-art techniques. The CRISPR/Cas9 system has great potential for efficiently generating mutants in non-traditional animal models and consequently to reinvigorate comparative behavioral neuroendocrinology. Here we describe the efficient generation of oxytocin receptor (Oxtr) mutant prairie voles (Microtus ochrogaster) using the CRISPR/Cas9 system, and describe initial behavioral phenotyping focusing on behaviors relevant to autism. Oxtr mutant male voles show no disruption in pup ultrasonic vocalization, anxiety as measured by the open field test, alloparental behavior, or sociability in the three chamber test. Mutants did however show a modest elevation in repetitive behavior in the marble burying test, and an impairment in preference for social novelty. The ability to efficiently generate targeted mutations in the prairie vole genome will greatly expand the utility of this model organism for discovering the genetic and circuit mechanisms underlying complex social behaviors, and serves as a proof of principle for expanding this approach to other non-traditional model organisms.

Keywords: Autism spectrum disorder; CRISPR/Cas9; Genome editing; Non-traditional models; Oxytocin receptor; Prairie voles; Social behavior; Social novelty preference.

Figure 1

Construction of sgRNAs targeting the prairie vole oxytocin receptor gene (Oxtr). (A) The target regions of each sgRNA in Oxtr. All sgRNAs were targeted against the 5’ region of the coding sequence (CDS). UTR = untranslated region. (B) Evaluation of cutting efficiencies by SSA assay in HEK293T cells (n = 3). The greater the number of EGFP positive cells, the greater the cutting efficiency, n.s = not significant. * = p<0.05. Data are presented as mean +/− Standard Error of the Mean (SEM). Scale bar = 150 μm.

Figure 2

Generation of Oxtr gene modified prairie voles by CRISPR/Cas. (A) Picture of vl, v2 and v3 mutant voles demonstrating their healthy appearance. (B) T7E1 assay for detection of indels induced by CRISPR/Cas. The presence of multiple bands is indicative of the introduction of indels.(C) Sequences of each mutant vole. Note the most voles had indels in both alleles of the Oxtr. Four mutant alleles in the v3 vole indicated that CRISPR might work at the 2-cell stage. Red letters = protospacer sequence; green letters = PAM; blue letters = insertion; magenta letters = conversion; hyphen = deletion.

Figure 3

Evidence for functional abnormality of OXTR in Oxtr^Δ60/Δ60 voles. (A) Representative data of the function of mutated Oxtr by TGFα shedding assay in vitro for vl, v4-1 and v4-2 voles (n = 3). (B) Genotypes of Oxtr^+/+, Oxtr^+/Δ60 and Oxtr^Δ60/Δ60 detected by PCR and agarose gel electrophoresis. (C) OXTR receptor autoradiography in wildtype (Oxtr^+/+) and Oxtr^Δ60/Δ60 Note the complete lack of signal in the Oxtr^Δ60/Δ60 brain. Cg, cingulate cortex; Acb, nucleus accumbens; Tu, tuberal region; Cl, claustrum; IC, insular cortex; M2, secondary motor cortex; LS, lateral septum; CPu, caudate putamen; FC, focus callosum; CeA, central amygdala; BLA, basolateral amygdala; VMH, ventromedial hypothalamus; VRe, ventral reuniens. Scale bar = 2mm.

Figure 4

Behavioral phenotype of male Oxtr^Δ60/Δ60 prairie voles. (A) The time course of behavioral tests. Each animal was tested in each test. (B) The duration and number of total calls in USV tests. (C) The time in center and total distance in OFT. (D) The number of buried marbles in MBT (E) The duration of licking and crouching and the relative number of retrieved pups in ABT. (F) Sociability and social novelty in TCT. * = p<0.05. n.s. = not significant. Data were presented as mean +/− SEM.