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Non-human primates as a model for human development

2021; Elsevier BV; Volume: 16; Issue: 5 Linguagem: Inglês

10.1016/j.stemcr.2021.03.021

2213-6711

Tomonori Nakamura, Kohei Fujiwara, Mitinori Saitou, Tomoyuki Tsukiyama,

Autism Spectrum Disorder Research

Human development has been studied for over a century, but the molecular mechanisms underlying human embryogenesis remain largely unknown due to technical difficulties and ethical issues. Accordingly, mice have been used as a model for mammalian development and studied extensively to infer human biology based on the conservation of fundamental processes between the two species. As research has progressed, however, species-specific differences in characteristics between rodents and primates have become apparent. Non-human primates (NHPs) have also been used for biomedical research, and are now attracting attention as a model for human development. Here, we summarize primate species from the evolutionary and genomic points of view. Then we review the current issues and progress in gene modification technology for NHPs. Finally, we discuss recent studies on the early embryogenesis of primates and future perspectives. Human development has been studied for over a century, but the molecular mechanisms underlying human embryogenesis remain largely unknown due to technical difficulties and ethical issues. Accordingly, mice have been used as a model for mammalian development and studied extensively to infer human biology based on the conservation of fundamental processes between the two species. As research has progressed, however, species-specific differences in characteristics between rodents and primates have become apparent. Non-human primates (NHPs) have also been used for biomedical research, and are now attracting attention as a model for human development. Here, we summarize primate species from the evolutionary and genomic points of view. Then we review the current issues and progress in gene modification technology for NHPs. Finally, we discuss recent studies on the early embryogenesis of primates and future perspectives. Rodents have been the predominant model organisms for mammalian biology so far. Mice in particular have numerous advantages that make them an excellent model animal, such as the ease of breeding, short generation time, and relatively large number of offspring. In addition, genome engineering technologies and pluripotent stem cell (PSC) technologies, which are essential for elucidating molecular mechanisms, have long been available in mice. As a result, many remarkable findings have been reported, some of which have contributed to our understanding of human biology as well as to the development of medicines. However, recent studies in rodents and humans have revealed that the gaps between the two species are larger than previously understood. Therefore, an animal model that is closer to humans is desired to infer human biology. In this sense, non-human primates (NHPs) are expected to be the best alternative. Both primates and rodents belong to the same subclade Euarchontoglires in clade Boreoeutheria, subclass Theria, class Mammalia. They are divided into the orders Primates and Rodents, which are thought to have diverged around 80 million years ago (mya) in the late Cretaceous period (Figure 1A). Primates now consist of more than 300 species, classified roughly into three major categories: New World monkeys/Platyrrhini, Old World monkeys/Catarrhini, and others. Human beings belong to the family Hominidae (also called the Great apes) in one clade of Old World monkeys/Catarrhini (Perelman et al., 2011Perelman P. Johnson W.E. Roos C. Seuanez H.N. Horvath J.E. Moreira M.A. Kessing B. Pontius J. Roelke M. Rumpler Y. et al.A molecular phylogeny of living primates.PLoS Genet. 2011; 7: e1001342Crossref PubMed Scopus (791) Google Scholar). Historically, four NHP species have been used for biomedical research with good success: chimpanzees, cynomolgus monkeys, rhesus monkeys, and marmosets (Johnsen et al., 2012Johnsen D.O. Johnson D.K. Whitney R.A. History of the use of nonhuman primates in biomedical research.in: Nonhuman Primates in Biomedical Research. 2012: 1-33Crossref Scopus (12) Google Scholar). Of these, the evolutionarily closest species to human beings (Homo sapiens) are chimpanzees (Pan troglodytes) in the Great apes, which also includes bonobos (Pan paniscus), gorillas (Gorilla gorilla), and orangutans (Pongo pygmaeus). Chimpanzees and bonobos belong to the same genus, Pan, and it is believed that they and humans diverged around 5–7 mya (Israfil et al., 2011Israfil H. Zehr S.M. Mootnick A.R. Ruvolo M. Steiper M.E. Unresolved molecular phylogenies of gibbons and siamangs (family: Hylobatidae) based on mitochondrial, Y-linked, and X-linked loci indicate a rapid Miocene radiation or sudden vicariance event.Mol. Phylogenet. Evol. 2011; 58: 447-455Crossref PubMed Scopus (31) Google Scholar; Prufer et al., 2012Prufer K. Munch K. Hellmann I. Akagi K. Miller J.R. Walenz B. Koren S. Sutton G. Kodira C. Winer R. et al.The bonobo genome compared with the chimpanzee and human genomes.Nature. 2012; 486: 527-531Crossref PubMed Scopus (294) Google Scholar) (Figure 1A), whereas gorillas and orangutans are thought to have divided from humans around 6–9 and 12–16 mya, respectively (Israfil et al., 2011Israfil H. Zehr S.M. Mootnick A.R. Ruvolo M. Steiper M.E. Unresolved molecular phylogenies of gibbons and siamangs (family: Hylobatidae) based on mitochondrial, Y-linked, and X-linked loci indicate a rapid Miocene radiation or sudden vicariance event.Mol. Phylogenet. Evol. 2011; 58: 447-455Crossref PubMed Scopus (31) Google Scholar; Locke et al., 2011Locke D.P. Hillier L.W. Warren W.C. Worley K.C. Nazareth L.V. Muzny D.M. Yang S.P. Wang Z. Chinwalla A.T. Minx P. et al.Comparative and demographic analysis of orang-utan genomes.Nature. 2011; 469: 529-533Crossref PubMed Scopus (348) Google Scholar; Scally et al., 2012Scally A. Dutheil J.Y. Hillier L.W. Jordan G.E. Goodhead I. Herrero J. Hobolth A. Lappalainen T. Mailund T. Marques-Bonet T. et al.Insights into hominid evolution from the gorilla genome sequence.Nature. 2012; 483: 169-175Crossref PubMed Scopus (430) Google Scholar) (Figure 1A). Currently, chimpanzees and the other Great apes are banned from use in invasive biomedical research in many countries (Johnsen et al., 2012Johnsen D.O. Johnson D.K. Whitney R.A. History of the use of nonhuman primates in biomedical research.in: Nonhuman Primates in Biomedical Research. 2012: 1-33Crossref Scopus (12) Google Scholar). And since chimpanzees are no longer permissible research models, the primates that are the next-most-closely related to humans are macaques. Accordingly, cynomolgus (Macaca fascicularis) and rhesus (Macaca mulatta) monkeys, belonging to macaques (genus Macaca) of the Old World monkeys/Catarrhini, have been the most extensively used NHPs for biomedical research. Currently, 23 macaque species are recognized as distinct animals, but mating between, for example, rhesus monkeys and Japanese monkeys, has been observed in Japan (Kawamoto et al., 2004Kawamoto Y. Hagihara K. Aizawa K. Finding of hybrid individuals between native Japanese macaques and introduced rhesus macaques in the Bousou Peninsula, Chiba, Japan.Primate Res. 2004; 20: 89-95Crossref Google Scholar) and this may imply that the differences among macaque species are so small as to be more like the differences among subspecies. NHPs belonging to the Old World monkeys/Catarrhini exist from Africa to the southern part of Eurasia and the Southeast Asian Islands, and macaques and the Great apes are thought to have branched 25–33 mya (Israfil et al., 2011Israfil H. Zehr S.M. Mootnick A.R. Ruvolo M. Steiper M.E. Unresolved molecular phylogenies of gibbons and siamangs (family: Hylobatidae) based on mitochondrial, Y-linked, and X-linked loci indicate a rapid Miocene radiation or sudden vicariance event.Mol. Phylogenet. Evol. 2011; 58: 447-455Crossref PubMed Scopus (31) Google Scholar; Locke et al., 2011Locke D.P. Hillier L.W. Warren W.C. Worley K.C. Nazareth L.V. Muzny D.M. Yang S.P. Wang Z. Chinwalla A.T. Minx P. et al.Comparative and demographic analysis of orang-utan genomes.Nature. 2011; 469: 529-533Crossref PubMed Scopus (348) Google Scholar; Rhesus Macaque Genome et al., 2007Rhesus Macaque Genome S. Analysis C. Gibbs R.A. Rogers J. Katze M.G. Bumgarner R. Weinstock G.M. Mardis E.R. Remington K.A. Strausberg R.L. et al.Evolutionary and biomedical insights from the rhesus macaque genome.Science. 2007; 316: 222-234Crossref PubMed Scopus (948) Google Scholar) (Figure 1A). Marmosets belong to the New World monkeys/Platyrrhini, which are mainly native to South America and are thought to have diverged from the Old World monkeys about 40 mya (Marmoset Genome and Analysis, 2014Marmoset Genome S. Analysis C. The common marmoset genome provides insight into primate biology and evolution.Nat. Genet. 2014; 46: 850-857Crossref PubMed Scopus (136) Google Scholar) (Figure 1A). Interestingly, while the other primates have evolved to increase their body size and lifespan, reduce the number of litters (basically becoming singletons), and prolong gestation, marmosets and their close relatives among New World monkeys have undergone reductions in body size from larger primate ancestors and evolved unique reproductive systems to include relatively short gestation and sexual maturation periods and to produce dizygotic twins sharing a single placental system (Figure 1B). As a result, the litters exchange hematopoietic stem cells in utero and have lifelong blood chimerism (Marmoset Genome and Analysis, 2014Marmoset Genome S. Analysis C. The common marmoset genome provides insight into primate biology and evolution.Nat. Genet. 2014; 46: 850-857Crossref PubMed Scopus (136) Google Scholar). Due to such unique characteristics, marmosets are also considered attractive research models and have been used for biomedical research as well. In the field of current biology, genome sequences are the essential pieces of information with which to understand many biological processes at the molecular level. So far, starting from the human genome in 2003 (International Human Genome Sequencing, 2004International Human Genome Sequencing C. Finishing the euchromatic sequence of the human genome.Nature. 2004; 431: 931-945Crossref PubMed Scopus (3138) Google Scholar), the chimpanzee genome was completed in 2005 (The Chimpanzee Sequencing and Analysis Consortium, 2005The Chimpanzee Sequencing and Analysis ConsortiumInitial sequence of the chimpanzee genome and comparison with the human genome.Nature. 2005; 437: 69-87Crossref PubMed Scopus (1623) Google Scholar), the rhesus monkey genome in 2007 (Rhesus Macaque Genome et al., 2007Rhesus Macaque Genome S. Analysis C. Gibbs R.A. Rogers J. Katze M.G. Bumgarner R. Weinstock G.M. Mardis E.R. Remington K.A. Strausberg R.L. et al.Evolutionary and biomedical insights from the rhesus macaque genome.Science. 2007; 316: 222-234Crossref PubMed Scopus (948) Google Scholar), the orangutan genome in 2011 (Locke et al., 2011Locke D.P. Hillier L.W. Warren W.C. Worley K.C. Nazareth L.V. Muzny D.M. Yang S.P. Wang Z. Chinwalla A.T. Minx P. et al.Comparative and demographic analysis of orang-utan genomes.Nature. 2011; 469: 529-533Crossref PubMed Scopus (348) Google Scholar), and the marmoset genome in 2015 (Marmoset Genome and Analysis, 2014Marmoset Genome S. Analysis C. The common marmoset genome provides insight into primate biology and evolution.Nat. Genet. 2014; 46: 850-857Crossref PubMed Scopus (136) Google Scholar; Sato et al., 2015Sato K. Kuroki Y. Kumita W. Fujiyama A. Toyoda A. Kawai J. Iriki A. Sasaki E. Okano H. Sakakibara Y. Resequencing of the common marmoset genome improves genome assemblies and gene-coding sequence analysis.Sci. Rep. 2015; 5: 16894Crossref PubMed Scopus (28) Google Scholar). Currently, the genomes of 18 primate species have been sequenced and are available in public databases. When the chimpanzee genome was announced in 2005, it revealed surprisingly that there was only a 1%–2% difference in alignable sequences between humans and chimpanzees, with more than 99.5% homology in the protein coding region (The Chimpanzee Sequencing and Analysis Consortium, 2005The Chimpanzee Sequencing and Analysis ConsortiumInitial sequence of the chimpanzee genome and comparison with the human genome.Nature. 2005; 437: 69-87Crossref PubMed Scopus (1623) Google Scholar). These results suggested that the evolution of the protein-coding sequences was not significant enough to explain the species differences among primates. On the other hand, nearly half of the primate genome consists of non-coding sequences and repetitive elements (International Human Genome Sequencing, 2004International Human Genome Sequencing C. Finishing the euchromatic sequence of the human genome.Nature. 2004; 431: 931-945Crossref PubMed Scopus (3138) Google Scholar). Of these, many families of endogenous retrotransposon are uniquely evolved in primate genomes. For example, the Alu element, which is a primate-specific family of short interspersed nuclear elements (SINEs), was specifically acquired in the Old World monkeys, and one endogenous retrovirus family, HERV was also acquired specifically only in humans (The Chimpanzee Sequencing and Analysis Consortium, 2005The Chimpanzee Sequencing and Analysis ConsortiumInitial sequence of the chimpanzee genome and comparison with the human genome.Nature. 2005; 437: 69-87Crossref PubMed Scopus (1623) Google Scholar; Rhesus Macaque Genome et al., 2007Rhesus Macaque Genome S. Analysis C. Gibbs R.A. Rogers J. Katze M.G. Bumgarner R. Weinstock G.M. Mardis E.R. Remington K.A. Strausberg R.L. et al.Evolutionary and biomedical insights from the rhesus macaque genome.Science. 2007; 316: 222-234Crossref PubMed Scopus (948) Google Scholar). These transposable elements make copies of themselves and transpose to other loci. Therefore, of course they are harmful to the host genome, and they are rapidly inactivated during the evolution of the host genome. Interestingly, however, such transposable elements can also drive the host evolution (Jacques et al., 2013Jacques P.E. Jeyakani J. Bourque G. The majority of primate-specific regulatory sequences are derived from transposable elements.Plos Genet. 2013; 9: e1003504Crossref PubMed Scopus (169) Google Scholar; Kunarso et al., 2010Kunarso G. Chia N.Y. Jeyakani J. Hwang C. Lu X. Chan Y.S. Ng H.H. Bourque G. Transposable elements have rewired the core regulatory network of human embryonic stem cells.Nat. Genet. 2010; 42: 631-634Crossref PubMed Scopus (469) Google Scholar). Bourque and colleagues investigated the binding sites of key transcription factors, POU5f1/Pou5f1 and NANOG/Nanog, in human and mouse embryonic stem cells (ESCs), and found that the binding patterns are markedly different, with only 5% of the regions being homologously occupied. Among the unconserved loci, ∼25% of binding sites were found in transposable elements, indicating that the transposable elements have the potential to dramatically change the transcriptional network (Kunarso et al., 2010Kunarso G. Chia N.Y. Jeyakani J. Hwang C. Lu X. Chan Y.S. Ng H.H. Bourque G. Transposable elements have rewired the core regulatory network of human embryonic stem cells.Nat. Genet. 2010; 42: 631-634Crossref PubMed Scopus (469) Google Scholar). They also investigated the marmoset and chimpanzee genomes and found that those potential transcription factor binding sites on transposable elements were highly species specific, suggesting that the transposable elements would contribute to the genome evolution through the formation of new transcriptional networks (Jacques et al., 2013Jacques P.E. Jeyakani J. Bourque G. The majority of primate-specific regulatory sequences are derived from transposable elements.Plos Genet. 2013; 9: e1003504Crossref PubMed Scopus (169) Google Scholar). It has recently become possible to identify such species-specific elements due to the development of long-read DNA sequencers. Most NHP genomes generated in the initial stage relied on guidance by the reference human genome. Accordingly, the NHP genomes have been somewhat "humanized." The advance of the long-read DNA sequencer has enabled us to overcome the problems on genome assembly and to identify structural variations among species (He et al., 2019He Y. Luo X. Zhou B. Hu T. Meng X. Audano P.A. Kronenberg Z.N. Eichler E.E. Jin J. Guo Y. et al.Long-read assembly of the Chinese rhesus macaque genome and identification of ape-specific structural variants.Nat. Commun. 2019; 10: 4233Crossref PubMed Scopus (18) Google Scholar; Kronenberg et al., 2018Kronenberg Z.N. Fiddes I.T. Gordon D. Murali S. Cantsilieris S. Meyerson O.S. Underwood J.G. Nelson B.J. Chaisson M.J.P. Dougherty M.L. et al.High-resolution comparative analysis of great ape genomes.Science. 2018; 360: eaar6343Crossref PubMed Scopus (129) Google Scholar). There are 17,000 ape-specific structural variants and many of them are located in enhancer regions. These data suggest that the species differences of phonotypes in primates may not be derived from the differences of protein types but rather from the differences in regulatory elements. Even though humans and rodents share basic biological processes, the species differences between them are not negligible and are becoming clearer along with advances in research. However, NHPs are often difficult to breed and prohibitively expensive (Johnsen et al., 2012Johnsen D.O. Johnson D.K. Whitney R.A. History of the use of nonhuman primates in biomedical research.in: Nonhuman Primates in Biomedical Research. 2012: 1-33Crossref Scopus (12) Google Scholar). Colony expansion by captive breeding has also been carried out, and this approach has been promoted by advances in reproductive technology such as hormone treatment approach in NHPs. However, captive breeding is still not easy, due not only to the long gestation and maturation periods, but also to the small number of pregnancies. Thus, NHPs have been used to model particularly serious and widespread diseases, including viral infections such as Ebola, HIV, and hepatitis B/C, which cannot be adequately replicated in mice, as well as for vaccine development and drug safety evaluation (Johnsen et al., 2012Johnsen D.O. Johnson D.K. Whitney R.A. History of the use of nonhuman primates in biomedical research.in: Nonhuman Primates in Biomedical Research. 2012: 1-33Crossref Scopus (12) Google Scholar). In 2020, NHPs were also used for research related to COVID-19 (Lu et al., 2020Lu S. Zhao Y. Yu W. Yang Y. Gao J. Wang J. Kuang D. Yang M. Yang J. Ma C. et al.Comparison of nonhuman primates identified the suitable model for COVID-19.Signal. Transduct. Target Ther. 2020; 5: 157Crossref PubMed Scopus (62) Google Scholar). They have also been widely used as a model of higher brain dysfunction that cannot be reproduced in mice. Accordingly, studies using NHPs have been primarily conducted in the areas of adult immunology, physiology, and neurophysiology. Thus, research for developmental biology has been very limited. However, these trends will be changed by the rapid progress in genome-editing technologies, such as the CRISPR-Cas9 system, and the development of stem cell biology based on the human PSCs, as will be discussed in greater detail in other sections, as well as single-cell analysis technologies. As mentioned above, the evaluation of mammalian gene function at the whole-body level had been limited to rodents for both technical and ethical reasons. Because there are no germline-transmittable ESCs or induced pluripotent stem cells (iPSCs) in non-rodent animals, including NHPs, the disruption of specific genes (knockout) and the introduction of genes into specific loci (knockin) was very difficult until the development of CRISPR-Cas9. Now that genome-editing technologies have been developing, however, there is increasing interest in the application of genome editing to various animals, including NHPs and humans. In research on human development, we can use surplus embryos after in vitro fertilization procedures with informed consent, but the supply of such embryos is limited and the permissibility of gene modification in human embryos is still under debate. At the moment, therefore, gene modification in NHPs is the best way to advance our understanding of human biology. Before the development of CRISPR-Cas9 editing, most reports on gene-modified NHPs involved transgenic (Tg) monkeys produced by viral vectors. Typically, in order to generate Tg mice, linearized vectors are injected into the pronuclei of zygotes. However, most mice generated by this method are genetically mosaic. Therefore, researchers need to generate multiple mouse lines and use them after the F1 generation (the generation after F0). When using mice, it is relatively easy to obtain a large number of lines and select them. However, for large animals such as NHPs, obtaining many lines is impractical in terms of time, cost, and labor because of the long sexual maturity and gestation periods. Thus, it is desirable to analyze the F0 generation (i.e., the first generation). Moreover, Tg mice that have insertions of full-length transgenes can only rarely be obtained in the F0 generation. Therefore, to improve the efficiency of the introduction of full-length transgenes, a viral vector system has been used to generate Tg animals in NHPs. The first successful generation of Tg animals in NHPs was described in 2001, when GFP-expressing vectors were introduced into rhesus monkey zygotes by retrovirus infection (Chan et al., 2001Chan A.W. Chong K.Y. Martinovich C. Simerly C. Schatten G. Transgenic monkeys produced by retroviral gene transfer into mature oocytes.Science. 2001; 291: 309-312Crossref PubMed Scopus (230) Google Scholar). Following this report, several technical improvements were achieved, such as the confirmation of germline transmission of lentiviral transgenes (Sasaki et al., 2009Sasaki E. Suemizu H. Shimada A. Hanazawa K. Oiwa R. Kamioka M. Tomioka I. Sotomaru Y. Hirakawa R. Eto T. et al.Generation of transgenic non-human primates with germline transmission.Nature. 2009; 459: 523-527Crossref PubMed Scopus (492) Google Scholar), analysis of differences in promoter types (Kim et al., 2007Kim S. Kim G.J. Miyoshi H. Moon S.H. Ahn S.E. Lee J.H. Lee H.J. Cha K.Y. Chung H.M. Efficiency of the elongation factor-1alpha promoter in mammalian embryonic stem cells using lentiviral gene delivery systems.Stem Cells Dev. 2007; 16: 537-545Crossref PubMed Scopus (45) Google Scholar; Seita et al., 2019Seita Y. Tsukiyama T. Azami T. Kobayashi K. Iwatani C. Tsuchiya H. Nakaya M. Tanabe H. Hitoshi S. Miyoshi H. et al.Comprehensive evaluation of ubiquitous promoters suitable for the generation of transgenic cynomolgus monkeys.Biol. Reprod. 2019; 100: 1440-1452Crossref PubMed Scopus (5) Google Scholar), analysis of viral injection timing (Kubisch et al., 2008Kubisch H.M. Gagliardi C. Romero D.G. Bunnell B.A. Ratterree M.S. Kinetics of pronuclear development and the effects of vector type and timing of injection on the efficiency of gene transfer into rhesus macaque embryos.Mol. Reprod. Dev. 2008; 75: 1505-1514Crossref PubMed Scopus (3) Google Scholar; Seita et al., 2016Seita Y. Tsukiyama T. Iwatani C. Tsuchiya H. Matsushita J. Azami T. Okahara J. Nakamura S. Hayashi Y. Hitoshi S. et al.Generation of transgenic cynomolgus monkeys that express green fluorescent protein throughout the whole body.Sci. Rep. 2016; 6: 24868Crossref PubMed Scopus (23) Google Scholar), and the application of different types of virus (Niu et al., 2010Niu Y. Yu Y. Bernat A. Yang S. He X. Guo X. Chen D. Chen Y. Ji S. Si W. et al.Transgenic rhesus monkeys produced by gene transfer into early-cleavage-stage embryos using a simian immunodeficiency virus-based vector.Proc. Natl. Acad. Sci. U S A. 2010; 107: 17663-17667Crossref PubMed Scopus (52) Google Scholar). Most of the published reports involving gene modification in NHPs were related to studies on disease modeling (Table S1). The pathological recapitulation of human disease is limited in mouse models because there are marked physiological differences between humans and mice. Indeed, many reports have demonstrated the superiority of monkey models over mouse models in this regard (Table S1). For example, duplications of MECP2-containing genomic segments cause a syndrome that shares core symptoms with autism spectrum disorders. It has been difficult to identify autism-like behaviors in the mouse model of MECP2 overexpression. In contrast, monkeys with MECP2 overexpression exhibit autism-like behaviors. These Tg monkeys show an increased frequency of repetitive circular locomotion, increased anxiety, reduced social interaction, and relatively weak cognitive phenotypes (Liu et al., 2016Liu Z. Li X. Zhang J.T. Cai Y.J. Cheng T.L. Chen C. Wang Y. Zhang C.C. Nie Y.H. Chen Z.F. et al.Autism-like behaviours and germline transmission in transgenic monkeys overexpressing MeCP2.Nature. 2016; 530: 98-102Crossref PubMed Scopus (154) Google Scholar). In addition, the phenotypic discrepancies between mice and humans are observed in several autosomal dominant diseases, such as autosomal dominant polycystic kidney disease (ADPKD). ADPKD, which is the most common hereditary kidney disease, is caused by PKD1 heterozygous mutations. However, heterozygous deletion of Pkd1 in mice rarely results in the formation of cysts until near the end of life. In contrast, PKD1 heterozygote monkeys exhibit cyst formation perinatally, as in humans (Tsukiyama et al., 2019Tsukiyama T. Kobayashi K. Nakaya M. Iwatani C. Seita Y. Tsuchiya H. Matsushita J. Kitajima K. Kawamoto I. Nakagawa T. et al.Monkeys mutant for PKD1 recapitulate human autosomal dominant polycystic kidney disease.Nat. Commun. 2019; 10: 5517Crossref PubMed Scopus (7) Google Scholar), highlighting the need for NHP models rather than mouse models. In humans, there are many autosomal dominant diseases and a study of such diseases requires selective production of heterozygotes. To produce heterozygotes selectively, a method for allele-specific targeting using polymorphism has been established (Tsukiyama et al., 2019Tsukiyama T. Kobayashi K. Nakaya M. Iwatani C. Seita Y. Tsuchiya H. Matsushita J. Kitajima K. Kawamoto I. Nakagawa T. et al.Monkeys mutant for PKD1 recapitulate human autosomal dominant polycystic kidney disease.Nat. Commun. 2019; 10: 5517Crossref PubMed Scopus (7) Google Scholar). In addition to this, to overcome the difficulties specific for NHPs, many other gene-modification methods have been developed, such as techniques for the specification of gene expression by tissue- or stage-specific promoters, the drug-inducible control of gene expression (Tomioka et al., 2017Tomioka I. Nogami N. Nakatani T. Owari K. Fujita N. Motohashi H. Takayama O. Takae K. Nagai Y. Seki K. Generation of transgenic marmosets using a tetracyclin-inducible transgene expression system as a neurodegenerative disease model.Biol. Reprod. 2017; 97: 772-780Crossref PubMed Scopus (17) Google Scholar; Tu et al., 2019Tu Z. Zhao H. Li B. Yan S. Wang L. Tang Y. Li Z. Bai D. Li C. Lin Y. et al.CRISPR/Cas9-mediated disruption of SHANK3 in monkey leads to drug-treatable autism-like symptoms.Hum. Mol. Genet. 2019; 28: 561-571Crossref PubMed Scopus (15) Google Scholar), reporter knockin into specific genes (Chu et al., 2019Chu C. Yang Z. Yang J. Yan L. Si C. Kang Y. Chen Z. Chen Y. Ji W. Niu Y. Homologous recombination-mediated targeted integration in monkey embryos using TALE nucleases.BMC Biotechnol. 2019; 19: 7Crossref PubMed Scopus (3) Google Scholar; Cui et al., 2018Cui Y. Niu Y. Zhou J. Chen Y. Cheng Y. Li S. Ai Z. Chu C. Wang H. Zheng B. et al.Generation of a precise Oct4-hrGFP knockin cynomolgus monkey model via CRISPR/Cas9-assisted homologous recombination.Cell Res. 2018; 28: 383-386Crossref PubMed Scopus (21) Google Scholar; Yao et al., 2017Yao X. Wang X. Hu X. Liu Z. Liu J. Zhou H. Shen X. Wei Y. Huang Z. Ying W. et al.Homology-mediated end joining-based targeted integration using CRISPR/Cas9.Cell Res. 2017; 27: 801-814Crossref PubMed Scopus (130) Google Scholar, Yao et al., 2018Yao X. Liu Z. Wang X. Wang Y. Nie Y.H. Lai L. Sun R. Shi L. Sun Q. Yang H. Generation of knock-in cynomolgus monkey via CRISPR/Cas9 editing.Cell Res. 2018; 28: 379-382Crossref PubMed Scopus (26) Google Scholar), and floxed allele knockin (Tsukiyama et al., 2019Tsukiyama T. Kobayashi K. Nakaya M. Iwatani C. Seita Y. Tsuchiya H. Matsushita J. Kitajima K. Kawamoto I. Nakagawa T. et al.Monkeys mutant for PKD1 recapitulate human autosomal dominant polycystic kidney disease.Nat. Commun. 2019; 10: 5517Crossref PubMed Scopus (7) Google Scholar). These technologies can be applied to developmental biology research to, for example, clarify the process by which germ cells and other cell lineages differentiate. Among the technical advances, the reduction of mosaicism is crucial for gene modification in NHPs. When analysis is performed with F0 animals, if any of the genetically modified individuals have mosaicism, expression of the phenotype may be hindered and phenotype analysis may become difficult. Several research groups have succeeded in reducing mosaicism in knockout monkey production (Midic et al., 2017Midic U. Hung P.H. Vincent K.A. Goheen B. Schupp P.G. Chen D.D. Bauer D.E. VandeVoort C.A. Latham K.E. Quantitative assessment of timing, efficiency, specificity and genetic mosaicism of CRISPR/Cas9-mediated gene editing of hemoglobin beta gene in rhesus monkey embryos.Hum. Mol. Genet. 2017; 26: 2678-2689Crossref PubMed Scopus (18) Google Scholar; Tsukiyama et al., 2019Tsukiyama T. Kobayashi K. Nakaya M. Iwatani C. Seita Y. Tsuchiya H. Matsushita J. Kitajima K. Kawamoto I. Nakagawa T. et al.Monkeys mutant for PKD1 recapitulate human autosomal dominant polycystic kidney disease.Nat. Commun. 2019; 10: 5517Crossref PubMed Scopus (7) Google Scholar; Tu et al., 2017Tu Z. Yang W. Yan S. Yin A. Gao J. Liu X. Zheng Y. Zheng J. Li Z. Yang S. et al.Promoting Cas9 degradation reduces mosaic mutations in non-