• non-human primate
• ovary
• reproduction
• reproductive engineering
• uterus

• ASD-like behavior
• animal models
• neurodevelopmental disorders
• scoring system

• Animals
• Disease Models, Animal
• Behavior, Animal
• Autism Spectrum Disorder
• Humans
• Rodentia
• Autistic Disorder/psychology
• Rats
• Mice

• assisted reproductive technologies
• cynomolgus monkey
• embryo transfer
• pregnancy outcome
• recipient

• Host–microbe interactions
• autism spectrum disorder
• early life
• gut-microbiota-brain axis
• hologenome
• neurodevelopmental disorders
• neuroimmune

• Humans
• Autism Spectrum Disorder/microbiology
• Autism Spectrum Disorder/etiology
• Female
• Pregnancy
• Exposome
• Gastrointestinal Microbiome
• Risk Factors
• Genetic Predisposition to Disease
• Animals
• Autistic Disorder/etiology
• Autistic Disorder/microbiology

• P30 ES030285 NIEHS NIH HHS
• R01 HD109095 NICHD NIH HHS
• R01 HD109780 NICHD NIH HHS

• African green monkey
• baboon
• blastoid
• bonobo
• chimera
• chimpanzee
• cynomolgus monkey
• embryonic stem cell
• gorilla
• induced pluripotent stem cell
• marmoset
• naive-state
• orangutan
• rhesus monkey

• Animals
• Chlorocebus aethiops
• Macaca mulatta
• Expeditions
• Pluripotent Stem Cells
• Cell Line
• Induced Pluripotent Stem Cells/metabolism
• Macaca fascicularis

• cell lineage
• heart stem cells
• stem-cell differentiation
• data processing

• Female
• Humans
• Animals
• Mice
• Placenta Growth Factor
• Endothelial Cells
• Myocardium
• Myocytes, Cardiac
• Cell Differentiation

• Vetenskapsrådet (Swedish Research Council)
• Hjärt-Lungfonden (Swedish Heart-Lung Foundation)
• Karolinska Institutet (Karolinska Institute)

• AAV-mediated gene delivery,
• CRISPR/Cas9,
• Functional reversal,
• Gene editing,
• Gene mutation,
• Gene therapy,
• Genetic engineered animal models,
• Neurodevelopmental Disorders
• Non-human primate models,
• Pharmacological targets,
• Translatability

• Animals
• Animals, Genetically Modified
• Cloning, Molecular
• Cloning, Organism/methods
• Gene Editing/methods
• Green Fluorescent Proteins/genetics
• Green Fluorescent Proteins/metabolism
• Macaca mulatta/genetics

• National Natural Science Foundation of China
• Natural Science Foundation of Yunnan Province
• National Key Research and Development Program of China Stem Cell and Translational Research

The foundation for investigating the mechanisms of human diseases is the establishment of animal models, which are also widely used in agricultural industry, pharmaceutical applications, and clinical research. However, small animals such as rodents, which have been extensively used to create disease models, do not often fully mimic the key pathological changes and/or important symptoms of human disease. As a result, there is an emerging need to establish suitable large animal models that can recapitulate important phenotypes of human diseases for investigating pathogenesis and developing effective therapeutics. However, traditional genetic modification technologies used in establishing small animal models are difficultly applied for generating large animal models of human diseases. This difficulty has been overcome to a great extent by the recent development of gene editing technology, especially the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9). In this review, we focus on the applications of CRISPR/Cas9 system to establishment of large animal models, including nonhuman primates, pigs, sheep, goats and dogs, for investigating disease pathogenesis and treatment. We also discuss the limitations of large animal models and possible solutions according to our current knowledge. Finally, we sum up the applications of the novel genome editing tool Base Editors (BEs) and its great potential for gene editing in large animals.

• National Key Research and Development Program of China
• National Natural Science Foundation of China

• Animals
• Disease Models, Animal
• Embryo, Mammalian
• Germ Cells
• Primates/genetics

• UG3 NS111688 NINDS NIH HHS
• K99 HD099154 NICHD NIH HHS
• P51 OD011106 NIH HHS
• R33 NS115102 NINDS NIH HHS
• R61 NS115102 NINDS NIH HHS

• hereditary eye diseases
• leber congenital amaurosis
• retinitis pigmentosa
• stargardt disease

• Animals
• Gene Editing
• Phenotype
• Primates/genetics
• Retina
• Retinal Diseases/genetics
• Retinal Diseases/therapy

Transcription activator-like effectors (TALEs) have been effectively used for targeted genome editing, transcriptional regulation, epigenetic modification, and locus-specific DNA imaging. However, with the advent of the clustered regularly interspaced short palindromic repeat/Cas9 system, an easy-to-use tool with the same function as TALEs, TALEs have recently been abandoned because of their complexity, time consumption, and difficult handling in common labs. Here, we described a degenerated codon-based TALE assembly system for simple, rapid, and efficient TALE assembly. TALE trimers with nonrepetitive DNA sequences were amplified by PCR and sequentially assembled via Gibson assembly. Our method is cost-effective, requires only commonly used basic molecular biology reagents, and takes only 2 h from target sequence analysis to completion. This simple, rapid, and lab-friendly TALE assembly method will restore the value of TALEs in DNA targeting.

• Gibson assembly
• degenerated codon
• transcription activator-like effectors
• trimer repeats

• Base Sequence
• CRISPR-Cas Systems
• Codon
• DNA/genetics
• Epigenesis, Genetic
• Gene Editing
• Gene Expression Regulation
• Genetic Engineering/methods
• Transcription Activator-Like Effectors/genetics

• biological techniques
• neuroscience
• neurology

• Animals
• Autografts
• CRISPR-Cas Systems
• Callithrix/genetics
• Embryo Culture Techniques
• Embryo Transfer/methods
• Female
• Fragile X Mental Retardation Protein/genetics
• Genetic Engineering/methods
• Models, Animal
• Mutation

• Japan Agency for Medical Research and Development

• China
• development biology
• model organism

Embryonic development is largely conserved among mammals. However, certain genes show divergent functions. By generating a transcriptional atlas containing >30,000 cells from post-implantation non-human primate embryos, we uncover that ISL1, a gene with a well-established role in cardiogenesis, controls a gene regulatory network in primate amnion. CRISPR/Cas9-targeting of ISL1 results in non-human primate embryos which do not yield viable offspring, demonstrating that ISL1 is critically required in primate embryogenesis. On a cellular level, mutant ISL1 embryos display a failure in mesoderm formation due to reduced BMP4 signaling from the amnion. Via loss of function and rescue studies in human embryonic stem cells we confirm a similar role of ISL1 in human in vitro derived amnion. This study highlights the importance of the amnion as a signaling center during primate mesoderm formation and demonstrates the potential of in vitro primate model systems to dissect the genetics of early human embryonic development.

Human and murine embryonic development has disparities, highlighting the need for primate systems. Here, the authors construct a post-implantation transcriptional atlas from non-human primate embryos and show ISL1 controls a gene regulatory network in the amnion required for mesoderm formation.

• MECP2 mutation monkey
• Rett syndrome
• diffusion tensor imaging
• graph theory
• microstructure

• CRISPR
• genetic modification
• moral status
• nonhuman primates
• novel beings
• personhood

• Autism spectrum disorders
• Disease model
• Gene-editing
• Nonhuman primate model

• autism
• autistic disorder
• cerebellum
• delay eyeblink conditioning
• motor learning
• social behavior

Most transgenic animals are generated using a genome-modified stem cell system and genome modification directly in embryos. Although this system is well-established in the development of transgenic animals, donor cell-derived transgenic animal production is inefficient in some cases. Especially in avian models such as chickens, the efficiency of transgenic animal production through primordial germ cells (PGCs) is highly variable compared with embryonic manipulation of mammalian species. Because germ cell and germline-competent stem cell-mediated systems that contain the transgene are enriched only at the upstream level during cell cultivation, the efficiency of transgenic animal production is unreliable. Therefore, we developed an in vivo selection model to enhance the efficiency of transgenic chicken production using microsomal glutathione-S-transferase II (MGSTII)-overexpressing PGCs that are resistant to the alkylating agent busulfan, which induces germ cell-specific cytotoxicity. Under in vitro conditions, MGSTII-tg PGCs were resistant to 1 μM busulfan, which was highly toxic to wild-type PGCs. In germline chimeric roosters, transgene-expressing germ cells were dominantly colonized in the recipient testes after busulfan exposure compared with non-treated germline chimera. In validation of germline transmission, donor PGC-derived progeny production efficiency was 94.68%, and the transgene production rate of heterozygous transgenic chickens was significantly increased in chickens that received 40 mg/kg busulfan (80.33–95.23%) compared with that of non-treated germline chimeras (51.18%). This system is expected to significantly improve the efficiency of generating transgenic chickens and other animal species by increasing the distribution of donor cells in adult testes.

• Culture of care
• Housing standards
• MRI
• Neuroimaging
• Primates
• Public engagement
• Welfare

• Access to Information/ethics
• Animal Welfare/ethics
• Animal Welfare/legislation & jurisprudence
• Animals
• Biomedical Research/ethics
• Biomedical Research/legislation & jurisprudence
• Committee Membership
• Humans
• Internationality
• Neurosciences/ethics
• Neurosciences/legislation & jurisprudence
• Primates

• 110157/Z/15/Z Wellcome Trust
• UF1 NS107668 NINDS NIH HHS
• R01 AG063090 NIA NIH HHS
• R01 EY017699 NEI NIH HHS
• Wellcome Trust
• R01 EY013692 NEI NIH HHS
• R01 MH064043 NIMH NIH HHS
• NC/K000802/1 National Centre for the Replacement, Refinement and Reduction of Animals in Research

• chimerism
• embryo
• genetic modification
• germ cell
• germline transmission
• hematopoietic stem cell
• marmoset monkey
• non-human primate
• transgenesis

• Animals
• Animals, Genetically Modified
• Callithrix
• Chimerism/embryology
• Female
• Green Fluorescent Proteins/metabolism
• Male

• 2010 Leibniz-Gemeinschaft

• CRISPR/Cas9
• Cardiovascular diseases
• Genome editing
• Induced pluripotent stem cells

• Brain/MINDS
• Callithrix jacchus
• common marmoset
• disease model
• genetic modification techniques
• genome editing technology
• neuroscience model

• Animals
• Animals, Genetically Modified
• Disease Models, Animal
• Gene Editing
• Humans
• Neurodevelopmental Disorders/genetics
• Neurodevelopmental Disorders/pathology
• Primates

• P50 MH094271 NIMH NIH HHS
• U01 MH114819 NIMH NIH HHS
• U24 OD026638 NIH HHS

• animal welfare
• ethics
• gene editing
• genetically modified
• macaque
• marmoset
• transgenesis
• transgenic

• NC3RS001 National Centre for the Replacement, Refinement and Reduction of Animals in Research

• CRISPR
• disease models
• genetic engineering
• nonhuman primate
• primates

• Animal Experimentation/ethics
• Animals
• Disease Models, Animal
• Mental Disorders/genetics
• Mental Disorders/physiopathology
• Nervous System Diseases/genetics
• Nervous System Diseases/physiopathology
• Neurosciences/ethics
• Neurosciences/methods
• Primates/genetics
• Primates/physiology

• P50 MH094271 NIMH NIH HHS
• P51 OD011107 NIH HHS
• RF1 AG061001 NIA NIH HHS
• R21 NS105437 NINDS NIH HHS
• U01 MH114819 NIMH NIH HHS
• R21 AG064448 NIA NIH HHS
• U24 OD026638 NIH HHS
• RF1 AG063837 NIA NIH HHS

• Drosophila
• Rett syndrome
• circadian rhythm
• monkey
• mouse
• sleep

Gene transcription is a crucial step in the sequence of molecular, synaptic, cellular, and systems mechanisms underlying learning and memory. Here, we review the experimental evidence demonstrating that alterations in the levels and functionality of the methylated DNA-binding transcriptional regulator MeCP2 are implicated in the learning and memory deficits present in mouse models of Rett syndrome and MECP2 duplication syndrome. The significant impact that MeCP2 has on gene transcription through a variety of mechanisms, combined with well-defined models of learning and memory, make MeCP2 an excellent candidate to exemplify the role of gene transcription in learning and memory. Together, these studies have strengthened the concept that precise control of activity-dependent gene transcription is a fundamental mechanism that ensures long-term adaptive behaviors necessary for the survival of individuals interacting with their congeners in an ever-changing environment.

• Gene Editing
• Genetic Diseases, Inborn/genetics
• Genetic Diseases, Inborn/therapy
• Germ Cells/metabolism
• Humans

One of the conventional methods to study the effects of gene mutations is that gene mutants are transfected into mammalian cells, and the dominant effects of gene mutants in the cells are examined. However, the result obtained using this method is not always satisfactory due to the interference of endogenous expression. Whether there is a better method to investigate the effects of gene mutations in cells remains to be examined. In the present study, a novel dual expression lentiviral vector was constructed using a shRNA-expressing lentiviral vector and combined techniques. Using this dual expression system, the vectors expressing both transcription factor IIA γ (TFIIAγ) shRNA and HA-TFIIAγ or its mutants were generated, and the effects of TFIIAγ gene mutations on transcription and protein–DNA interaction were investigated. We show that the transfection of the vector expressing TFIIAγ shRNA and HA-TFIIAγ fusion gene was able to silence the expression of endogenous TFIIAγ gene but not affect that of exogenous HA-TFIIAγ fusion gene in either transiently transfected cells or stable cell lines. Mutations in the conservative domain between AA₆₂ and AA₆₉ in TFIIAγ inhibit the activities of promoters and endogenous gene expression, and reduce TFIIAγ binding to AdML core promoter compared with wild-type (WT) TFIIAγ. ChIP-qPCR data suggest that the TFIIAγ N63A mutant inhibits insulin-like growth factor 2 (IGF2) transcription by reducing the recruitments of TFIIAγ, polymerase II (Pol II), TATA box-binding protein (TBP), and TBP associated factor 1 (250 kDa) (TAF1) at its promoter. Our study provides a novel method that is used to investigate the effects of gene mutations at the cellular level.

• cellular level
• dual expression vector
• gene mutation effect
• gene transcription
• method

• Clustered Regularly Interspaced Palindromic Repeat
• environment
• ethics
• genome editing
• human health

• Animals
• CRISPR-Cas Systems
• Gene Editing/ethics
• Gene Editing/veterinary

• Stichting voor de Technische Wetenschappen
• John Templeton Foundation
• British Academy

• agriculture
• disease model
• genome editing
• large animal
• xenotransplantation

• MCPH1
• brain development
• cognition
• human evolution
• neoteny
• transgenic monkey

• animal models
• behavioral biomarkers
• cognitive impairment
• translational research
• treatments

• animal models
• neuroscience
• nonhuman primates

Rare diseases are very difficult to study mechanistically and to develop therapies for because of the scarcity of patients. Here, the rare neuro-metabolic disorder Rett syndrome (RTT) is discussed as a prototype for precision medicine, demonstrating how mouse models have led to an understanding of the development of symptoms. RTT is caused by mutations in the X-linked gene methyl-CpG-binding protein 2 (MECP2). Mecp2-mutant mice are being used in preclinical studies that target the MECP2 gene directly, or its downstream pathways. Importantly, this work may improve the health of RTT patients. Clinical presentation may vary widely among individuals based on their mutation, but also because of the degree of X chromosome inactivation and the presence of modifier genes. Because it is a complex disorder involving many organ systems, it is likely that recovery of RTT patients will involve a combination of treatments. Precision medicine is warranted to provide the best efficacy to individually treat RTT patients.

• Alcohol
• CRISPR/Cas
• Genetically engineered animals
• Genome editing

• Alcoholism/etiology
• Alcoholism/genetics
• Animals
• Animals, Genetically Modified
• CRISPR-Cas Systems/genetics
• Gene Editing
• Mice
• Rats
• Transcription Activator-Like Effector Nucleases/physiology

• R01 AA030257 NIAAA NIH HHS
• R37 AA010422 NIAAA NIH HHS
• U01 AA020889 NIAAA NIH HHS

Cloning of macaque monkeys by somatic cell nucleus transfer (SCNT) allows the generation of monkeys with uniform genetic backgrounds that are useful for the development of non-human primate models of human diseases. Here, we report the feasibility of this approach by SCNT of fibroblasts from a macaque monkey (Macaca fascicularis), in which a core circadian transcription factor BMAL1 was knocked out by clustered regularly interspaced short palindromic repeat/Cas9 gene editing (see accompanying paper). Out of 325 SCNT embryos transferred into 65 surrogate monkeys, we cloned five macaque monkeys with BMAL1 mutations in both alleles without mosaicism, with nuclear genes identical to that of the fibroblast donor monkey. Further peripheral blood mRNA analysis confirmed the complete absence of the wild-type BMAL1 transcript. This study demonstrates that the SCNT approach could be used to generate cloned monkeys from fibroblasts of a young adult monkeys and paves the way for the development of macaque monkey disease models with uniform genetic backgrounds.