Symposia

Symposia Cutting-edge Genome Editing Technology and Next Generation Disease Model Animals／最先端のゲノム編集技術と次世代疾患モデル動物研究

3S1-1 Opportunities and challenges in animal model innovation in CRISPR-era Tomomi Aida^1,2,3,4 ¹McGovern Institute for Brain Research, Massachusetts Institute of Technology，²Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard，³Lab Mol Neurosci. MRI, TMDU，⁴Lab Recombinant Animals, MRI, TMDU With the rapid emergence of new genome editing technologies like a CRISPR, it is becoming increasingly feasible and much easier to manipulate genomes in a wide range of species including primates. This CRISPR revolution opens a new avenue for brain research, disease modeling, preclinical study, and gene therapy. Here, I review the latest achievements of CRISPR technologies and subsequent opportunities with a specific focus on the animal models. I will also discuss the current limitations and challenges in this rapidly changing field. The goal of this session will be to share the latest opportunities and challenges in the ongoing innovation of animal models with the community, and stimulate our idea to accelerate and move brain research forward in CRISPR-era.		3S1-2 CRISPR-mediated genome editing in animals Tomoji Mashimo^1,2 ¹Inst Exp Anim Sci, Grad Sch Med, Osaka Univ，²GERDC, Grad Sch Med, Osaka Univ The CRISPR/Cas9 system is a powerful tool for generating genetically modified animals. We have so far reported the efficient generation of ssODN-mediated knock-ins (KIs) in rats, i.e., SNP exchange, 19-bp DNA integration, and elimination of 7 k-bp ERV sequences (Yoshimi et al. Nat Commun 2014). We have also shown that co-injection of two gRNAs as “scissors” to cut the genomic DNA and the donor DNA, and two short ssODNs as “paste”, resulting in efficient KI of plasmid DNAs, including a 200-kb BAC, at the targeted site (Yoshimi et al. Nat Commun 2016). In addition, we have reported zygote electroporation instead of microinjection is an efficient genome editing in mice and rats (Kaneko et al. Sci Rep 2014). Here we will show the use of long single-stranded DNA (lssDNA) as a targeting donor, which are synthesized by a method using nicking endonucleases. Zygote electroporation of lssDNAs with CRISPR/Cas9 provides efficient KI of GFP sequences at the targeted site. The lssDNAs also enables the quick generation of floxed alleles in zygotes. The high efficiency of this approach, termed CLICK (CRISPR with lssDNA inducing conditional knockout alleles), provides homozygous knock-ins in oocytes expressing tissue-specific Cre, which allows the one-step generation of conditional knockouts in founder (F0) mice. The zygote electroporation, CLICK, is applicable to any target site with any donor sequences, thus facilitating easy and flexible genome engineering in mice, rats, and rabbits.

3S1-3 Deciphering the mechanisms of development and diseases of the gyrencephalic brain using carnivore ferrets Hiroshi Kawasaki Dept Med Neurosci, Grad Sch Med, Kanazawa Univ The ferret, a carnivore, has the large and complicated brain than the rodent. For example, ferrets have folds on the cerebral cortex (i.e. the gyrus and the sulcus), whereas mice do not have. To investigate the molecular mechanisms underlying development and diseases related to the brain structures unique to higher mammals, we have developed a genetic manipulation technique for the cerebral cortex of ferrets using in utero electroporation. Genes-of-interest can be expressed in the ferret cortex rapidly and efficiently. Using our technique, we examined the mechanisms underlying cortical folding. We focused on the Tbr2 transcription factor, which is expressed in neural progenitors of the subventricular zone (SVZ). When Tbr2 was suppressed, we found that the number of SVZ progenitors was markedly reduced and that cortical folding was significantly impaired. Interestingly, upper layers were more reduced than lower layers, suggesting the ratio between upper layers and lower layers is important for cortical folding. In addition, we found that introduction of fibroblast growth factors resulted in polymicrogyria, in which additional folds were made. Pax6-positive cells and Tbr2-positive cells were increased, and upper layers were preferentially increased. Our findings provide in vivo data about the mechanisms of cortical folding in gyrencephalic mammals. The ferret cerebral cortex is useful for investigating the mechanisms underlying development and diseases of brain structures unique to higher mammals.		3S1-4 Genetically-Modified Cynomolgus Monkeys for Human Disease Modeling Masatsugu Ema¹，Tomoyuki Tsukiyama¹，Kenichi Kobayashi²，Koji Fukuda³，Teppei Iwakiri³，Hiroyuki Izumi³，Akihiro Kawauchi²，Seiji Hitoshi⁴，Erika Sasaki⁵，Yasunari Seita¹ ¹Department of Stem Cells and Human Disease Models Research Center for Animal Life Science Shiga University of Medical Science，²Department of Urology, Shiga University of Medical Science，³Shin Nippon Biomedical Laboratories, Ltd，⁴Department of Physiology, Shiga University of Medical Science，⁵Central Institute for Experimental Animals Mice are valuable for human disease modeling, and many genetically-modified mice have been created over the past 40 years by DNA microinjection into pronuclear embryos, by using homologous recombination in embryonic stem cells and by CRISPR/Cas9 system. However, mice poorly recapitulate some human diseases such as Parkinson’s disease and Alzheimer’s disease. Therefore, there is a need to establish animal models to recapitulate human diseases more faithfully. In this regard, nonhuman primates (NHPs) are considered one of the most valuable animal models because NHPs are closer to humans in organ size and anatomical structure. Accordingly, they have a greater potential to recapitulate human diseases, although the difficulty of genetic manipulation is a major issue in creating the disease models. Here, I summarize our recent trials in transgenic and genome editing approaches with a NHP, the cynomolgus monkey (Macaca fascicularis) for human disease modeling.

3S1-5 Modeling Human Psychiatric/Neurological Disorders using Transgenic technology in Non-human Primates Hideyuki Okano Department of Physiology, Keio University School of Medicine There is increasing interest in common marmosets (Callithrix jacchus) in the neuroscience and disease modeling, since they exhibit some enriched human-like traits less exhibited by Old World monkeys (Miller et al., Neuron, 2016). For faithful modeling human psychiatric and neurological diseases in vivo, we developed lentivirus-mediated transgenic techniques with germline transmission (Sasaki et al., Nature, 2009) and genome editing (Sato et al., Cell Stem Cell, 2016). In the present talk, I wish to mention our recent data of generation of transgenic marmoset models of neurodegenererative diseases, including Parkinson disease which overexpressed the mutant form of alpha-synuclein (A30P) using lentiviral vector. Currently, we are developing a technique for creating knockout marmosets using zinc finger nuclease (ZFN) and Transcription Activator-Like Effector Nuclease (TALEN) technology, based on the genomic sequence data (Sato et al., Sci Rep, 2015). By using this technique, we could successfully generate interleukin-2 receptor subunit gamma (IL2RG)-deficient marmosets (Sato et al., Cell Stem Cell, 2016). Together with the development of cognitive information for marmoset brain analysis, innovative MRI imaging technology and marmoset genetic analysis tools, we created and are analyzing MECP2 mutant marmosets suitable for research on Rett syndrome, a neurodevelopmental disorder showing autistic behavior, to understand the pathogenesis, and to contribute to new therapeutic strategies to treat Rett syndrome. In the present talk, I will talk about the detailed phenotypic analysis of MECP2 mutant marmosets and Brain Mapping Projects in Japan using genetically-modified marmoset (Okano et al. Neuron, 2016). (Supported by Brain/MINDS from A-MED)