CRISPR/Cas9 enables the targeting of genes in zygotes; however, efficient approaches to create loxP-flanked (floxed) alleles remain elusive. Here, we show that the electroporation of Cas9, two gRNAs, and long single-stranded DNA (lssDNA) into zygotes, termed CLICK (CRISPR with lssDNA inducing conditional knockout alleles), enables the quick generation of floxed alleles in mice and rats. The high efficiency of CLICK provides homozygous knock-ins in oocytes carrying tissue-specific Cre, which allows the one-step generation of conditional knockouts in founder (F0) mice.

GABAergic dysfunctions have been implicated in the pathogenesis of schizophrenia, especially the associated cognitive impairments. The GABA synthetic enzyme glutamate decarboxylase 67-kDa isoform (GAD67) encoded by the GAD1 gene is downregulated in the brains of patients with schizophrenia. Furthermore, a patient with schizophrenia harboring a homozygous mutation of GAD1 has recently been discovered. However, it remains unclear whether loss of function of GAD1 leads to the symptoms observed in schizophrenia, including cognitive impairment. One of the obstacles faced in experimental studies to address this issue is the perinatal lethality of Gad1 knockout (KO) mice, which precluded characterization at the adult stage. In the present study, we successfully generated Gad1 KO rats using CRISPR/Cas9 genome editing technology. Surprisingly, 33% of Gad1 KO rats survived to adulthood and could be subjected to further characterization. The GABA concentration in the Gad1 KO cerebrum was reduced to ~52% of the level in wild-type rats. Gad1 KO rats exhibited impairments in both spatial reference and working memory without affecting adult neurogenesis in the hippocampus. In addition, Gad1 KO rats showed a wide range of behavioral alterations, such as enhanced sensitivity to an NMDA receptor antagonist, hypoactivity in a novel environment, and decreased preference for social novelty. Taken together, the results suggest that Gad1 KO rats could provide a novel model covering not only cognitive deficits but also other aspects of the disorder. Furthermore, the present study teaches an important lesson: differences between species should be considered when developing animal models of human diseases.

Immunodeficient animals are valuable models for the engraftment of exogenous tissues; they are widely used in many fields, including the creation of humanized animal models, as well as regenerative medicine and oncology. Compared with mice, laboratory rats have a larger body size and can more easily undergo transplantation of various tissues and organs. Considering the absence of high-quality resources of immunodeficient rats, we used the CRISPR/Cas9 genome editing system to knock out the interleukin-2 receptor gamma chain gene (Il2rg) in F344/Jcl rats—alone or together with recombination activating gene 2 (Rag2)—to create a high-quality bioresource that researchers can freely use: severe combined immunodeficiency (SCID) rats. We selected one founder rat with frame-shift mutations in both Il2rg (5-bp del) and Rag2 ([1-bp del+2-bp ins]/[7-bp del+2-bp ins]), then conducted mating to establish a line of immunodeficient rats. The immunodeficiency phenotype was preliminarily confirmed by the presence of severe thymic hypoplasia in Il2rg-single knockout (sKO) and Il2rg/Rag2-double knockout (dKO) rats. Assessment of blood cell counts in peripheral blood showed that the white blood cell count was significantly decreased in sKO and dKO rats, while the red blood cell count was unaffected. The decrease in white blood cell count was mainly caused by a decrease in lymphocytes. Furthermore, analyses of lymphocyte populations via flow cytometry showed that the numbers of B cells (CD3^- CD45⁺) and natural killer cells (CD3^- CD161⁺) were markedly reduced in both knockout rats. In contrast, T cells were markedly reduced but showed slightly different results between sKO and dKO rats. Notably, our immunodeficient rats do not exhibit growth retardation or gametogenesis defects. This high-quality SCID rat resource is now managed by the National BioResource Project in Japan. Our SCID rat model has been used in various research fields, demonstrating its importance as a bioresource.

Placebo analgesia is caused by inactive treatment, implicating endogenous brain function involvement. However, the neurobiological basis remains unclear. In this study, we found that μ-opioid signals in the medial prefrontal cortex (mPFC) activate the descending pain inhibitory system to initiate placebo analgesia in neuropathic pain rats. Chemogenetic manipulation demonstrated that specific activation of μ-opioid receptor–positive (MOR ⁺ ) neurons in the mPFC or suppression of the mPFC–ventrolateral periaqueductal gray (vlPAG) circuit inhibited placebo analgesia in rats. MOR ⁺ neurons in the mPFC are monosynaptically connected and directly inhibit layer V pyramidal neurons that project to the vlPAG via GABA _A receptors. Thus, intrinsic opioid signaling in the mPFC disinhibits excitatory outflow to the vlPAG by suppressing MOR ⁺ neurons, leading to descending pain inhibitory system activation that initiates placebo analgesia. Our results shed light on the fundamental neurobiological mechanism of the placebo effect that maximizes therapeutic efficacy and reduces adverse drug effects in medical practice.

The GABAergic system is thought to play an important role in the control of cognition and emotion, such as fear, and is related to the pathophysiology of psychiatric disorders. For example, the expression of the 67‐kDa isoform of glutamate decarboxylase (GAD67), a GABA‐producing enzyme, is downregulated in the postmortem brains of patients with major depressive disorder and schizophrenia. However, knocking out the Gad1 gene, which encodes GAD67, is lethal in mice, and thus, the association between Gad1 and cognitive/emotional functions is unclear. We recently developed Gad1 knockout rats and found that some of them can grow into adulthood. Here, we performed fear‐conditioning tests in adult Gad1 knockout rats to assess the impact of the loss of Gad1 on fear‐related behaviors and the formation of fear memory. In a protocol assessing both cued and contextual memory, Gad1 knockout rats showed a partial antiphase pattern of freezing during training and significantly excessive freezing during the contextual test compared with wild‐type rats. However, Gad1 knockout rats did not show any synchronous increase in freezing with auditory tones in the cued test. On the other hand, in a contextual memory specialized protocol, Gad1 knockout rats exhibited comparable freezing behavior to wild‐type rats, while their fear extinction was markedly impaired. These results suggest that GABA synthesis by GAD67 has differential roles in cued and contextual fear memory.