Ex utero monkey embryogenesis from blastocyst to early organogenesis.

The third and fourth weeks of gestation in primates are marked by several developmental milestones, including gastrulation and the formation of organ primordia. However, our understanding of this period is limited due to restricted access to in vivo embryos. To address this gap, we developed an embedded 3D culture system that allows for the extended ex utero culture of cynomolgus monkey embryos for up to 25 days post-fertilization. Morphological, histological, and single-cell RNA-sequencing analyses demonstrate that ex utero cultured monkey embryos largely recapitulated key events of in vivo development. With this platform, we were able to delineate lineage trajectories and genetic programs involved in neural induction, lateral plate mesoderm differentiation, yolk sac hematopoiesis, primitive gut, and primordial germ-cell-like cell development in monkeys. Our embedded 3D culture system provides a robust and reproducible platform for growing monkey embryos from blastocysts to early organogenesis and studying primate embryogenesis ex utero.

Modeling Rett Syndrome Using TALEN-Edited MECP2 Mutant Cynomolgus Monkeys.

Gene-editing technologies have made it feasible to create nonhuman primate models for human genetic disorders. Here, we report detailed genotypes and phenotypes of TALEN-edited MECP2 mutant cynomolgus monkeys serving as a model for a neurodevelopmental disorder, Rett syndrome (RTT), which is caused by loss-of-function mutations in the human MECP2 gene. Male mutant monkeys were embryonic lethal, reiterating that RTT is a disease of females. Through a battery of behavioral analyses, including primate-unique eye-tracking tests, in combination with brain imaging via MRI, we found a series of physiological, behavioral, and structural abnormalities resembling clinical manifestations of RTT. Moreover, blood transcriptome profiling revealed that mutant monkeys resembled RTT patients in immune gene dysregulation. Taken together, the stark similarity in phenotype and/or endophenotype between monkeys and patients suggested that gene-edited RTT founder monkeys would be of value for disease mechanistic studies as well as development of potential therapeutic interventions for RTT.

Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos.

Monkeys serve as important model species for studying human diseases and developing therapeutic strategies, yet the application of monkeys in biomedical researches has been significantly hindered by the difficulties in producing animals genetically modified at the desired target sites. Here, we first applied the CRISPR/Cas9 system, a versatile tool for editing the genes of different organisms, to target monkey genomes. By coinjection of Cas9 mRNA and sgRNAs into one-cell-stage embryos, we successfully achieve precise gene targeting in cynomolgus monkeys. We also show that this system enables simultaneous disruption of two target genes (Ppar-γ and Rag1) in one step, and no off-target mutagenesis was detected by comprehensive analysis. Thus, coinjection of one-cell-stage embryos with Cas9 mRNA and sgRNAs is an efficient and reliable approach for gene-modified cynomolgus monkey generation.

IFI16 promotes the progression of clear cell renal cell carcinoma through the IL6/PI3K/AKT axis.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is a common disease in the urinary system, with a high incidence and poor prognosis in advanced stages. Although γ-interferon-inducible protein 16 (IFI16) has been reported to play a role in various tumors, its involvement in ccRCC remains poorly documented, and the molecular mechanisms are not yet clear. METHODS: We conducted bioinformatics analysis to study the expression of IFI16 in ccRCC using public databases. Additionally, we analyzed and validated clinical specimens that we collected. Subsequently, we explored the impact of IFI16 on ccRCC cell proliferation, migration, and invasion through in vitro and in vivo experiments. Furthermore, we predicted downstream molecules and pathways using transcriptome analysis and confirmed them through follow-up experimental validation. RESULTS: IFI16 was significantly upregulated in ccRCC tissue and correlated with poor patient prognosis. In vitro, IFI16 promoted ccRCC cell proliferation, migration, and invasion, while in vivo, it facilitated subcutaneous tumor growth and the formation of lung metastatic foci. Knocking down IFI16 suppressed its oncogenic function. At the molecular level, IFI16 promoted the transcription and translation of IL6, subsequently activating the PI3K/AKT signaling pathway and inducing epithelial-mesenchymal transition (EMT). CONCLUSION: IFI16 induced EMT through the IL6/PI3K/AKT axis, promoting the progression of ccRCC.

Neural Stem Cells Transplanted into Rhesus Monkey Cortical Traumatic Brain Injury Can Survive and Differentiate into Neurons.

Cortical traumatic brain injury (TBI) is a major cause of cognitive impairment accompanied by motor and behavioral deficits, and there is no effective treatment strategy in the clinic. Cell transplantation is a promising therapeutic strategy, and it is necessary to verify the survival and differentiation of cells after transplantation in large animal models like rhesus monkeys. In this study, we transplanted neural stem cells (NSCs) and simultaneously injected basic fibroblast growth factor/epidermal growth factor (bFGF/EGF) into the cortex (visual and sensory cortices) of rhesus monkeys with superficial TBI. The results showed that the transplanted NSCs did not enter the cerebrospinal fluid (CSF) and were confined to the transplantation site for at least one year. The transplanted NSCs differentiated into mature neurons that formed synaptic connections with host neurons, but glial scar formation between the graft and the host tissue did not occur. This study is the first to explore the repairing effect of transplanting NSCs into the superficial cerebral cortex of rhesus monkeys after TBI, and the results show the ability of NSCs to survive long-term and differentiate into neurons, demonstrating the potential of NSC transplantation for cortical TBI.

Longitudinal brain atlases of early developing cynomolgus macaques from birth to 48 months of age.

Longitudinal brain imaging atlases with densely sampled time-points and ancillary anatomical information are of fundamental importance in studying early developmental characteristics of human and non-human primate brains during infancy, which feature extremely dynamic imaging appearance, brain shape and size. However, for non-human primates, which are highly valuable animal models for understanding human brains, the existing brain atlases are mainly developed based on adults or adolescents, denoting a notable lack of temporally densely-sampled atlases covering the dynamic early brain development. To fill this critical gap, in this paper, we construct a comprehensive set of longitudinal brain atlases and associated tissue probability maps (gray matter, white matter, and cerebrospinal fluid) with totally 12 time-points from birth to 4 years of age (i.e., 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36, and 48 months of age) based on 175 longitudinal structural MRI scans from 39 typically-developing cynomolgus macaques, by leveraging state-of-the-art computational techniques tailored for early developing brains. Furthermore, to facilitate region-based analysis using our atlases, we also provide two popular hierarchy parcellations, i.e., cortical hierarchy maps (6 levels) and subcortical hierarchy maps (6 levels), on our longitudinal macaque brain atlases. These early developing atlases, which have the densest time-points during infancy (to the best of our knowledge), will greatly facilitate the studies of macaque brain development.

Secretory type II cGMP-dependent protein kinase blocks activation of PDGFRß via Ser254 in gastric cancer cells.

The study of secretory protein kinase is an emergent research field in recent years. The secretion phenomenon of type II cGMP-dependent protein kinase (PKG II) was found in our latest research and our previous study confirmed that PKG II inhibited platelet-derived growth factor receptor ß (PDGFRß) activation induced by platelet-derived growth factor BB (PDGF-BB) within the gastric cancer cells. Thus, this study was designed to investigated effect of secretory PKG II on PDGFRß. Transwell assay and CCK8 assay indicated that secretory PKG II reversed PDGF-BB-induced cell migration, invasion, and proliferation. Immunoprecipitation, GST pull down and Western blotting results showed that secretory PKG II combined with extracellular domains of PDGFRß and phosphorylated it, and thereby inhibited PDGF-BB-induced activation of PDGFRß, and downstream PI3K/Akt and MAPK/ERK pathways. Mutation at Ser254 of PDGFRß to alanine abolished the above inhibitory effects of secretory PKG II on PDGFRß, indicating that Ser254 was the specific site phosphorylated by secretory PKG II. In conclusion, secretory PKG II inhibited PDGFRß activation via Ser254 site.

White Matter Structural and Network Topological Changes Underlying the Behavioral Phenotype of MECP2 Mutant Monkeys.

To explore the brain structural basis underlying the behavioral abnormalities associated with Rett syndrome (RTT), we carried out detailed longitudinal noninvasive magnetic resonance imaging analyses of RTT monkey models created by gene-editing, from weaning, through adolescence, till sexual maturation. Here, we report abnormal developmental dynamics of brain white matter (WM) microstructures and network topological organizations via diffusion tensor imaging. Specifically, disrupted WM microstructural integrity was observed at 9 months, but recovered thereafter, whereas WM network topological properties showed persistent abnormal dynamics from 9 to 37 months. Changes in the WM microstructure and WM network topology were correlated well with RTT-associated behavioral abnormalities including sleep latency, environmental exploration, and conflict encounters. Deleterious and protracted early WM myelination process likely lead to abnormal synaptic pruning, resulting in poor functional segregations. Together, this study provides initial evidence for changes in WM microstructure and network topological organization, which may underlie the neuro-patho-etilogy of RTT.

Celastrol acts synergistically with afatinib to suppress non-small cell lung cancer cell proliferation by inducing paraptosis.

Non-small cell lung cancer (NSCLC) with wild-type epidermal growth factor receptor (EGFR) is intrinsic resistance to EGFR-tyrosine kinase inhibitors (TKIs), such as afatinib. Celastrol, a natural compound with antitumor activity, was reported to induce paraptosis in cancer cells. In this study, intrinsic EGFR-TKI-resistant NSCLC cell lines H23 (EGFR wild-type and KRAS mutation) and H292 (EGFR wild-type and overexpression) were used to test whether celastrol could overcome primary afatinib resistance through paraptosis induction. The synergistic effect of celastrol and afatinib on survival inhibition of the NSCLC cells was evaluated by CCK-8 assay and isobologram analysis. The paraptosis and its modulation were assessed by light and electron microscopy, Western blot analysis, and immunofluorescence. Xenografts models were established to investigate the inhibitory effect of celastrol plus afatinib on the growth of the NSCLC tumors in vivo. Results showed that celastrol acted synergistically with afatinib to suppress the survival of H23 and H292 cells by inducing paraptosis characterized by extensive cytoplasmic vacuolation. This process was independent of apoptosis and not associated with autophagy induction. Afatinib plus celastrol-induced cytoplasmic vacuolation was preceded by endoplasmic reticulum stress and unfolded protein response. Accumulation of intracellular reactive oxygen species and mitochondrial Ca2+ overload may be initiating factors of celastrol/afatinib-induced paraptosis and subsequent cell death. Furthermore, Celastrol and afatinib synergistically suppressed the growth of H23 cell xenograft tumors in vivo. The data indicate that a combination of afatinib and celastrol may be a promising therapeutic strategy to surmount intrinsic afatinib resistance in NSCLC cells.

[MRI/TRUS cognitive fusion combined with 12-core systematic transperineal prostate biopsy for the diagnosis of clinically significant prostate cancer: A report of 208 cases].

OBJECTIVE: To investigate the detection rate and complications of magnetic resonance imaging / transrectal ultrasonography (MRI/TRUS) cognitive fusion combined with 12-core systematic transperineal prostate biopsy (TPPB) in the diagnosis of clinically significant PCa (CS-PCa). METHODS: This retrospective study included 208 patients undergoing first-time MRI/TRUS cognitive fusion combined with 12-core systematic TPPB from June 2015 to May 2019. The patients, aged 54－85 (67.6 ± 7.8) years, all received digital rectal examination, PSA detection, TRUS and prostate multiparametric MRI (mpMRI) before biopsy. We analyzed the mpMRI images, identified and marked the suspected signal areas, repeated TRUS for further observation of the prostate, conducted cognitive fusion based on the mpMRI images and determined the target before 12-core systematic TPPB and subjecting the samples obtained to pathological examination. RESULTS: Of the 208 patients, 112 were diagnosed with CS-PCa (no case with tPSA < 4 µg/L, 21 cases with 4 µg/L ≤ tPSA < 10 µg/L, 47 cases with 10 µg/L ≤ tPSA < 20 µg/L, 40 cases with 20 µg/L ≤ tPSA < 100 µg/L, and 4 cases with tPSA ≥ 100 µg/L), 85 with BPH, 8 with chronic prostatitis, 2 with atypical prostatic hyperplasia, and 1 with prostatic intraepithelial neoplasia. Systemic inflammatory response syndrome occurred in 3 and gross hematuria and/or bloody stool in 12 cases after biopsy, which were all cured by anti-infection and hemostasis treatment. CONCLUSIONS: MRI/TRUS cognitive fusion combined with 12-core systematic transperineal prostate biopsy can improve the detection rate of the initial diagnosis of clinically significant PCa with a low incidence of controllable complications.

PARP inhibitor resistance: the underlying mechanisms and clinical implications.

Due to the DNA repair defect, BRCA1/2 deficient tumor cells are more sensitive to PARP inhibitors (PARPi) through the mechanism of synthetic lethality. At present, several PAPRi targeting poly (ADP-ribose) polymerase (PARP) have been approved for ovarian cancer and breast cancer indications. However, PARPi resistance is ubiquitous in clinic. More than 40% BRCA1/2-deficient patients fail to respond to PARPi. In addition, lots of patients acquire PARPi resistance with prolonged oral administration of PARPi. Homologous recombination repair deficient (HRD), as an essential prerequisite of synthetic lethality, plays a vital role in killing tumor cells. Therefore, Homologous recombination repair restoration (HRR) becomes the predominant reason of PARPi resistance. Recently, it was reported that DNA replication fork protection also contributed to PARPi resistance in BRCA1/2-deficient cells and patients. Moreover, various factors, such as reversion mutations, epigenetic modification, restoration of ADP-ribosylation (PARylation) and pharmacological alteration lead to PARPi resistance as well. In this review, we reviewed the underlying mechanisms of PARP inhibitor resistance in detail and summarized the potential strategies to overcome PARPi resistance and increase PARPi sensitivity.

No off-target mutations in functional genome regions of a CRISPR/Cas9-generated monkey model of muscular dystrophy.

CRISPR/Cas9 is now widely used in biomedical research and has great potential for clinical applications. However, the safety and efficacy of this gene-editing technique are significant issues. Recent reports on mouse models and human cells have raised concerns that off-target mutations could hamper applying the CRISPR technology in patients. The high similarities of nonhuman primates to humans in genome content and organization, genetic diversity, physiology, and cognitive abilities have made these animals ideal experimental models for understanding human diseases and developing therapeutics. Off-target mutations of CRISPR/Cas9 have been analyzed in previous studies of nonhuman primates, but no report has investigated genome-wide off-target effects in living monkeys. Here, we used rhesus monkeys in which a genetic disorder mimicking Duchenne muscular dystrophy had previously been produced with CRISPR/Cas9. Using whole-genome sequencing to comprehensively assess on- and off-target mutations in these animals, we found that CRISPR/Cas9-based gene editing is active on the expected genomic sites without producing off-target modifications in other functional regions of the genome. These findings suggest that the CRISPR/Cas9 technique could be relatively safe and effective in modeling genetic disease in nonhuman primates and in future therapeutic research of human diseases.

Type II cGMP-dependent protein kinase phosphorylates EGFR at threonine 669 and thereby inhibits its activation.

Our previous study demonstrated that type II cGMP-dependent protein kinase (PKG II) inhibited epidermal growth factor (EGF) induced tyrosine phosphorylation/activation of the EGF receptor (EGFR). This paper was designed to investigate the mechanism of the inhibition of PKG II on EGFR activation. Gastric cancer cells HGC-27 and AGS were infected with an adenoviral vector encoding the cDNA of PKG II (Ad-PKG II) to overexpress PKG II and treated with 8-(4-chlorophenylthio) guanosine-3',5'-cyclic monophosphate (8-pCPT-cGMP) to activate the kinase. Co-immunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC) assay were performed to detect the interaction between PKG II and EGFR. Western blotting, mass spectrometry (MS) and site mutagenesis were performed to detect the PKG II-specific phosphorylation site on EGFR. The results showed that in living COS-7 cells, which were infected with Ad-PKG II and treated with 8-pCPT-cGMP, there was an interaction between PKG II and EGFR. The results also showed that PKG II caused threonine 669 (T669) phosphorylation of EGFR in HGC-27 and AGS cells infected with Ad-PKG II and treated with 8-pCPT-cGMP, and then inhibited the activation of EGFR. When T669 of EGFR was mutated to alanine, the inhibitory effect of PKG II on the activation of EGFR was eradicated. These findings suggested a PKG II-specific phosphorylation site on EGFR, and might be beneficial to illuminate the anti-tumor role of PKG II.

Homologous recombination-mediated targeted integration in monkey embryos using TALE nucleases.

BACKGROUND: Non-human primate (NHP) models can closely mimic human physiological functions and are therefore highly valuable in biomedical research. Genome editing is now developing rapidly due to the precision and efficiency offered by engineered site-specific endonuclease-based systems, such as transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) system. It has been demonstrated that these programmable nucleases can introduce genetic changes in embryos from many species including NHPs. In 2014, we reported the first genetic editing of macaques using TALENs and CRISPR/Cas9. Subsequently, we characterized the phenotype of a methyl CpG binding protein 2 (MECP2)-mutant cynomolgus monkey model of Rett syndrome generated using the TALEN approach. These efforts not only accelerated the advance of modeling genetic diseases in NHPs, but also encouraged us to develop specific gene knock-in monkeys. In this study, we assess the possibility of homologous recombination (HR)-mediated gene replacement using TALENs in monkeys, and generate preimplantation embryos carrying an EmGFP fluorescent reporter constructed in the OCT4 gene. RESULT: We assembled a pair of TALENs specific to the first exon of the OCT4 gene and constructed a donor vector consisting of the homology arms cloned from the monkey genome DNA, flanking an EmGFP cassette. Next, we co-injected the TALENs-coding plasmid and donor plasmid into the cytoplasm of 122 zygotes 6-8 h after fertilization. Sequencing and immunofluorescence revealed that the OCT4-EmGFP knock-in allele had been successfully generated by TALENs-mediated HR at an efficiency of 11.3% (7 out of 62) or 11.1% (1 out of 9), respectively, in monkey embryos. CONCLUSION: We have successfully, for the first time, obtained OCT4-EmGFP knock-in monkey embryos via HR mediated by TALENs. Our results suggest that gene targeting through TALEN-assisted HR is a useful approach to introduce precise genetic modification in NHPs.

PKG II effectively reversed EGF-induced protein expression alterations in human gastric cancer cell lines.

Epidermal growth factor receptor (EGFR) plays an important role in gastric cancer (GC) progression. Our previous data demonstrated that type II cGMP-dependent protein kinase (PKG II) could block the EGF-EGFR axis as well as down-stream signaling pathways, for example, MAPK, PI3 K, and PLC in GC cells. However, the exact mechanisms of PKG II against cancer remain unclear. Therefore, the present work was to address the above question. Human GC cell line AGS was infected with adenoviral construct encoding cDNA of PKG II (Ad-PKG II) to up-regulate PKG II and then treated with 8-pCPT-cGMP. Two-dimensional electrophoresis (2-DE) was used to analyze the changes of protein expression in the cells. The results showed that 17 proteins had more than twofold changes in EGF-treated group compared with control. However, Ad-PKG II could effectively reversed the changes. Furthermore, far upstream element-binding protein 1 (FUBP1) and MarvelD3 were chosen and PKG II activation reversed EGF/EGFR-induced up-regulation of FUBP1 and downregulation of MarvelD3, respectively. MarvelD3 silence effectively abolished the inhibitory effect of PKG II on EGF-triggered migration. These data indicated that the inhibitory effect of PKG II partially was associated with MarvelD3.

PKG II inhibits PDGF-BB triggered biological activities by phosphorylating PDGFRß in gastric cancer cells.

Previous studies revealed that type II cGMP-dependent protein kinase G (PKG II) could inhibit the activation of epidermal growth factor receptor (EGFR) which is a widely investigated RTK. PDGFR belongs to family of receptor tyrosine kinases (RTKs) too. However, the effect of PKG II on PDGFR activation is not clear yet. This study investigated potential regulatory effect of PKG II on activation of PDGFRß and the downstream signaling transductions in gastric cancer. The results from CCK8 assay and Transwell assay indicated that PDGF-BB induced cell proliferation and migration. Activated PKG II reversed the above variations caused by PDGF-BB. Immunoprecipitation and Western blotting results showed that PKG II combined with PDGFRß and phosphorylated this receptor, and thereby inhibited PDGF-BB induced activation of PDGFRß, and MAPK/ERK and PI3K/Akt mediated signal transduction pathways. Based on the prediction by phosphorylation site software, Ser643 and Ser712 were mutated to alanine respectively which prevented phosphorylation at these sites. Mutation at Ser712 abolished the inhibitory function of PKG II on PDGFRß activation but mutation of Ser643 had no such an effect, indicating that Ser712 was PKG II-specific phosphorylating site of PDGFRß. In conclusion, PKG II inhibited PDGFRß activation in gastric cancer via phosphorylating Ser712 of this RTK.

Cyclic Guanosine Monophosphate (cGMP)-Dependent Protein Kinase II Blocks Epidermal Growth Factor (EGF)/Epidermal Growth Factor Receptor (EGFR)-Induced Biological Effects on Osteosarcoma Cells.

BACKGROUND The present work was performed to detect the potential inhibitory effect of cyclic guanosine monophosphate (cGMP)-dependent protein kinase II (PKG II) on epidermal growth factor (EGF) receptor-induced biological activity and related signal cascades in osteosarcoma cells. MATERIAL AND METHODS We transfected the osteosarcoma MG-63 cell line with an adenoviral vector encoding PKG II cDNA (Ad-PKGII) and incubated the transfected cells with 250 µM 8-pCPT-cGMP to activate the PKG II. We stimulated the MG-63 cells with100 ng/ml EGF, and then detected their proliferation using a CCK-8 assay. Transwell assay was used to examine MG-63 cell migration; and Western blot analysis was used to detect expression of matrix metalloproteinase 9 (MMP-9) and activation of ERK and Akt. RESULTS Stimulating cells by 100 ng/ml EGF promoted MG-63 cell proliferation and migration, ERK and Akt phosphorylation, and MMP-9 expression. These effects of EGF were inhibited in MG-63 cells infected with Ad-PKGII and incubated with 8-pCPT-cGMP. CONCLUSIONS Our results demonstrate that Ad-PKGII infection significantly inhibited EGF-induced proliferation and migration, as well as the associated-signal cascades; which indicates that PKG II might be a potential anti-cancer factor.

Protein Kinases Type II (PKG II) Combined with L-Arginine Significantly Ameliorated Xenograft Tumor Development: Is L-Arginine a Potential Alternative in PKG II Activation?

BACKGROUND The mammalian cyclic guanosine monophosphate (cGMP)-dependent protein kinases type II (PKG II) plays critical physiological or pathological functions in different tissues. However, the biological effects of PKG II are dependent on cGMP. Published data indicated that L-arginine (L-Arg) promoted NO production, NO can activate soluble guanylate cyclase (sGC), and catalyzes guanosine triphosphate (GTP) into cGMP, which suggested L-Arg could activate PKG II. Therefore, the present work was performed to address: (i) whether L-Arg could be a potential alternative in PKG II activation, and (ii) whether L-Arg also contributes to PKG II against cancer. MATERIAL AND METHODS Nude BALB/c mice were inoculated with human MCF-7, HepG2, and SW480 cell lines via subcutaneous (s.c.) injecting. After 7 days of inoculation, Ad-PKG II was injected into the cancer tissues every 4 days, and the next day 10 µmol/mouse L-Arg was administered. Western blotting and immunohistochemistry were used to assess protein expression. RESULTS Our results demonstrated that L-Arg significantly activated PKG II and effectively ameliorated xenograft tumor development through inhibiting cancer growth, angiogenesis, and metastasis, which was partially dependent on blocking of epidermal growth factor receptor (EGFR) activity, as well as downstream signaling pathways such as Erk1/2. CONCLUSIONS Our results provide an exciting new insight: L-Arg is a potential alternative to PKG II activation.

CRISPR/Cas9-mediated Dax1 knockout in the monkey recapitulates human AHC-HH.

Mutations in the DAX1 locus cause X-linked adrenal hypoplasia congenita (AHC) and hypogonadotropic hypogonadism (HH), which manifest with primary adrenal insufficiency and incomplete or absent sexual maturation, respectively. The associated defects in spermatogenesis can range from spermatogenic arrest to Sertoli cell only syndrome. Conclusions from Dax1 knockout mouse models provide only limited insight into AHC/HH disease mechanisms, because mouse models exhibit more extensive abnormalities in testicular development, including disorganized and incompletely formed testis cords with decreased number of peritubular myoid cells and male-to-female sex reversal. We previously reported successful clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome targeting in cynomolgus monkeys. Here, we describe a male fetal monkey in which targeted genome editing using CRISPR/Cas9 produced Dax1-null mutations in most somatic tissues and in the gonads. This DAX1-deficient monkey displayed defects in adrenal gland development and abnormal testis architecture with small cords, expanded blood vessels and extensive fibrosis. Sertoli cell formation was not affected. This phenotype strongly resembles findings in human patients with AHC-HH caused by mutations in DAX1. We further detected upregulation of Wnt/ß-catenin-VEGF signaling in the fetal Dax1-deficient testis, suggesting abnormal activation of signaling pathways in the absence of DAX1 as one mechanism of AHC-HH. Our study reveals novel insight into the role of DAX1 in HH and provides proof-of-principle for the generation of monkey models of human disease via CRISPR/Cas9-mediated gene targeting.