Huntington's disease (HD) is characterized by preferential loss of the medium spiny neurons in the striatum. Using CRISPR/Cas9 and somatic nuclear transfer technology, we established a knockin (KI) pig model of HD that endogenously expresses full-length mutant huntingtin (HTT). By breeding this HD pig model, we have successfully obtained F1 and F2 generation KI pigs. Characterization of founder and F1 KI pigs shows consistent movement, behavioral abnormalities, and early death, which are germline transmittable. More importantly, brains of HD KI pig display striking and selective degeneration of striatal medium spiny neurons. Thus, using a large animal model of HD, we demonstrate for the first time that overt and selective neurodegeneration seen in HD patients can be recapitulated by endogenously expressed mutant proteins in large mammals, a finding that also underscores the importance of using large mammals to investigate the pathogenesis of neurodegenerative diseases and their therapeutics.

CRISPR/Cas9 has been used to genetically modify genomes in a variety of species, including non-human primates. Unfortunately, this new technology does cause mosaic mutations, and we do not yet know whether such mutations can functionally disrupt the targeted gene or cause the pathology seen in human disease. Addressing these issues is necessary if we are to generate large animal models of human diseases using CRISPR/Cas9. Here we used CRISPR/Cas9 to target the monkey dystrophin gene to create mutations that lead to Duchenne muscular dystrophy (DMD), a recessive X-linked form of muscular dystrophy. Examination of the relative targeting rate revealed that Crispr/Cas9 targeting could lead to mosaic mutations in up to 87% of the dystrophin alleles in monkey muscle. Moreover, CRISPR/Cas9 induced mutations in both male and female monkeys, with the markedly depleted dystrophin and muscle degeneration seen in early DMD. Our findings indicate that CRISPR/Cas9 can efficiently generate monkey models of human diseases, regardless of inheritance patterns. The presence of degenerated muscle cells in newborn Cas9-targeted monkeys suggests that therapeutic interventions at the early disease stage may be effective at alleviating the myopathy.

CRISPR-Cas9 is a powerful new tool for genome editing, but this technique creates mosaic mutations that affect the efficiency and precision of its ability to edit the genome. Reducing mosaic mutations is particularly important for gene therapy and precision genome editing. Although the mechanisms underlying the CRSIPR/Cas9-mediated mosaic mutations remain elusive, the prolonged expression and activity of Cas9 in embryos could contribute to mosaicism in DNA mutations. Here we report that tagging Cas9 with ubiquitin-proteasomal degradation signals can facilitate the degradation of Cas9 in non-human primate embryos. Using embryo-splitting approach, we found that shortening the half-life of Cas9 in fertilized zygotes reduces mosaic mutations and increases its ability to modify genomes in non-human primate embryos. Also, injection of modified Cas9 in one-cell embryos leads to live monkeys with the targeted gene modifications. Our findings suggest that modifying Cas9 activity can be an effective strategy to enhance precision genome editing.CRISPR-Cas9 combines the type II bacterial clustered, regularly interspaced, palindromic repeats (CRISPR)-associated (Cas) system and a single guide RNA (gRNA), which targets Cas9 to genomic regions complementary to the gRNA 1 . Double-stranded DNA breaks at the targeted locus are generated by Cas9 digestion and repaired by non-homologous end-joining (NHEJ) or homology-directed repair (HDR), resulting in indel mutations [2][3]

Animal models are extremely valuable to help us understand the pathogenesis of neurodegenerative disorders and to find treatments for them. Since large animals are more like humans than rodents, they make good models to identify the important pathological events that may be seen in humans but not in small animals; large animals are also very important for validating effective treatments or confirming therapeutic targets. Due to the lack of embryonic stem cell lines from large animals, it has been difficult to use traditional gene targeting technology to establish large animal models of neurodegenerative diseases. Recently, CRISPR/Cas9 was used successfully to genetically modify genomes in various species. Here we discuss the use of CRISPR/Cas9 technology to establish large animal models that can more faithfully mimic human neurodegenerative diseases.

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Despite the substantial progress made in identifying genetic defects in autism spectrum disorder (ASD), the etiology for majority of ASD individuals remains elusive. Maternal exposure to valproic acid (VPA), a commonly prescribed antiepileptic drug during pregnancy in human, has long been considered a risk factor to contribute to ASD susceptibility in offspring from epidemiological studies in humans. The similar exposures in murine models have provided tentative evidence to support the finding from human epidemiology. However, the apparent difference between rodent and human poses a significant challenge to extrapolate the findings from rodent models to humans. Here we report for the first time the neurodevelopmental and behavioral outcomes of maternal VPA exposure in non-human primates. Monkey offspring from the early maternal VPA exposure have significantly reduced NeuN-positive mature neurons in prefrontal cortex (PFC) and cerebellum and the Ki67-positive proliferating neuronal precursors in the cerebellar external granular layer, but increased GFAP-positive astrocytes in PFC. Transcriptome analyses revealed that maternal VPA exposure disrupted the expression of genes associated with neurodevelopment in embryonic brain in offspring. VPA-exposed juvenile offspring have variable presentations of impaired social interaction, pronounced stereotypies, and more attention on nonsocial stimuli by eye tracking analysis. Our findings in non-human primates provide the best evidence so far to support causal link between maternal VPA exposure and neurodevelopmental defects and ASD susceptibility in humans.

Parkinson's disease (PD) is an age-dependent neurodegenerative disease that often occurs in those over age 60. Although rodents and small animals have been used widely to model PD and investigate its pathology, their short life span makes it difficult to assess the aging-related pathology that is likely to occur in PD patient brains. Here, we used brain tissues from rhesus monkeys at 2-3, 7-8, and Ͼ15 years of age to examine the expression of Parkin, PINK1, and ␣-synuclein, which are known to cause PD via loss-or gain-of-function mechanisms. We found that ␣-synuclein is increased in the older monkey brains, whereas Parkin and PINK1 are decreased or remain unchanged. Because of the gain of toxicity of ␣-synuclein, we performed stereotaxic injection of lentiviral vectors expressing mutant ␣-synuclein (A53T) into the substantia nigra of monkeys and found that aging also increases the accumulation of A53T in neurites and its associated neuropathology. A53T also causes more extensive reactive astrocytes and axonal degeneration in monkey brain than in mouse brain. Using monkey brain tissues, we found that A53T interacts with neurofascin, an adhesion molecule involved in axon subcellular targeting and neurite outgrowth. Aged monkey brain tissues show an increased interaction of neurofascin with A53T. Overexpression of A53T causes neuritic toxicity in cultured neuronal cells, which can be attenuated by transfected neurofascin. These findings from nonhuman primate brains reveal age-dependent pathological and molecular changes that could contribute to the age-dependent neuropathology in PD.