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CRISPR/Cas9-engineered Gad1 elimination in rats leads to complex behavioral changes: implications for schizophrenia.

ABSTRACT: 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.

SUBMITTER: Fujihara K

PROVIDER: S-EPMC7723991 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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CRISPR/Cas9-engineered Gad1 elimination in rats leads to complex behavioral changes: implications for schizophrenia.

Fujihara Kazuyuki K Yamada Kazuo K Ichitani Yukio Y Kakizaki Toshikazu T Jiang Weiru W Miyata Shigeo S Suto Takashi T Kato Daiki D Saito Shigeru S Watanabe Masahiko M Kajita Yuki Y Ohshiro Tomokazu T Mushiake Hajime H Miyasaka Yoshiki Y Mashimo Tomoji T Yasuda Hiroki H Yanagawa Yuchio Y

Translational psychiatry 20201208 1

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 s ...[more]

PMID: 33293518

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Project description:BackgroundA barrier to HIV-1 cure rests in the persistence of proviral DNA in infected CD4+ leukocytes. The high HIV-1 mutation rate leads to viral diversity, immune evasion, and consequent antiretroviral drug resistance. While CRISPR-spCas9 can eliminate latent proviral DNA, its efficacy is limited by HIV strain diversity and precision target cell delivery.MethodsA library of guide RNAs (gRNAs) designed to disrupt five HIV-1 exons (tat1-2/rev1-2/gp41) was constructed. The gRNAs were derived from a conseensus sequence of the transcriptional regulator tat from 4004 HIV-1 strains. Efficacy was affirmed by gRNA cell entry through transfection, electroporation, or by lentivirus or lipid nanoparticle (LNP) delivery. Treated cells were evaluated for viral excision by monitoring HIV-1 DNA, RNA, protein, and progeny virus levels.FindingsVirus was reduced in all transmitted founder strains by 82 and 94% after CRISPR TatDE transfection or lentivirus treatments, respectively. No recorded off-target cleavages were detected. Electroporation of TatDE ribonucleoprotein and delivery of LNP TatDE gRNA and spCas9 mRNA to latently infected cells resulted in up to 100% viral excision. Protection against HIV-1-challenge or induction of virus during latent infection, in primary or transformed CD4+ T cells or monocytes was achieved. We propose that multi-exon gRNA TatDE disruption delivered by LNPs enables translation for animal and human testing.InterpretationThese results provide "proof of concept' for CRISPR gRNA treatments for HIV-1 elimination. The absence of full-length viral DNA by LNP delivery paired with undetectable off-target affirms the importance of payload delivery for effective viral gene editing.FundingThe work was supported by the University of Nebraska Foundation, including donations from the Carol Swarts, M.D. Emerging Neuroscience Research Laboratory, the Margaret R. Larson Professorship, and individual donor support from the Frances and Louie Blumkin Foundation and from Harriet Singer. The research received support from National Institutes of Health grants T32 NS105594, 5R01MH121402, 1R01Al158160, R01 DA054535, PO1 DA028555, R01 NS126089, R01 NS36126, PO1 MH64570, P30 MH062261, and 2R01 NS034239.

Project description:BackgroundDesminopathy is a clinically heterogeneous muscle disease caused by over 60 different mutations in desmin. The most common mutation with a clinical phenotype in humans is an exchange of arginine to proline at position 350 of desmin leading to p.R350P. We created the first CRISPR-Cas9 engineered rat model for a muscle disease by mirroring the R350P mutation in humans.MethodsUsing CRISPR-Cas9 technology, Des c.1045-1046 (AGG > CCG) was introduced into exon 6 of the rat genome causing p.R349P. The genotype of each animal was confirmed via quantitative PCR. Six male rats with a mutation in desmin (n = 6) between the age of 120-150 days and an equal number of wild type littermates (n = 6) were used for experiments. Maximal plantar flexion force was measured in vivo and combined with the collection of muscle weights, immunoblotting, and histological analysis. In addition to the baseline phenotyping, we performed a synergist ablation study in the same animals.ResultsWe found a difference in the number of central nuclei between desmin mutants (1 ± 0.4%) and wild type littermates (0.2 ± 0.1%; P < 0.05). While muscle weights did not differ, we found the levels of many structural proteins to be altered in mutant animals. Dystrophin and syntrophin were increased 54% and 45% in desmin mutants, respectively (P < 0.05). Dysferlin and Annexin A2, proteins associated with membrane repair, were increased two-fold and 32%, respectively, in mutants (P < 0.05). Synergist ablation caused similar increases in muscle weight between mutant and wild type animals, but changes in fibre diameter revealed that fibre hypertrophy in desmin mutants was hampered compared with wild type animals (P < 0.05).ConclusionsWe created a novel animal model for desminopathy that will be a useful tool in furthering our understanding of the disease. While mutant animals at an age corresponding to a preclinical age in humans show no macroscopic differences, microscopic and molecular changes are already present. Future studies should aim to further decipher those biological changes that precede the clinical progression of disease and test therapeutic approaches to delay disease progression.

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CRISPR/Cas9-engineered Gad1 elimination in rats leads to complex behavioral changes: implications for schizophrenia.

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CRISPR/Cas9-engineered Gad1 elimination in rats leads to complex behavioral changes: implications for schizophrenia.

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