Genome editing tools such as the clustered regularly interspaced short palindromic repeat （CRISPR）-associated system （Cas） have been widely used to modify genes in model systems including animal zygotes and human ...Genome editing tools such as the clustered regularly interspaced short palindromic repeat （CRISPR）-associated system （Cas） have been widely used to modify genes in model systems including animal zygotes and human cells, and hold tremendous promise for both basic research and clinical applications. To date, a serious knowledge gap remains in our understanding of DNA repair mechanisms in human early embryos, and in the efficiency and potential off-target effects of using technologies such as CRISPR/Cas9 in human pre-implantation embryos. In this report, we used tripronuclear （3PN） zygotes to further investigate CRISPR/Cas9-mediated gene editing in human cells. We found that CRISPR/Cas9 could effectively cleave the endogenous β-globin gene （HBB）. However, the efficiency of homologous recombination directed repair （HDR） of HBB was low and the edited embryos were mosaic. Off-target cleavage was also apparent in these 3PN zygotes as revealed by the T7E1 assay and whole-exome sequencing. Furthermore, the endogenous delta-globin gene （HBD）, which is homologous to HBB, competed with exogenous donor oligos to act as the repair template, leading to untoward mutations. Our data also indicated that repair of the HBB locus in these embryos occurred preferentially through the non-crossover HDR pathway. Taken together, our work highlights the pressing need to further improve the fidelity and specificity of the CRISPR/Cas9 platform, a prerequisite for any clinical applications of CRSIPR/Cas9-mediated editing.展开更多
油菜是我国重要的油料作物,常年种植面积约1亿亩,每年可生产约450万t菜籽油,占国内植物油总消费量的19.7%。与发达国家相比,我国油菜产业主要问题是产量低、品质差,年进口油菜籽约500万t。油菜基因组测序的完成,极大地推动了油菜育种行...油菜是我国重要的油料作物,常年种植面积约1亿亩,每年可生产约450万t菜籽油,占国内植物油总消费量的19.7%。与发达国家相比,我国油菜产业主要问题是产量低、品质差,年进口油菜籽约500万t。油菜基因组测序的完成,极大地推动了油菜育种行业的科研工作。据统计(Web of Science检索),2017年与油菜育种相关的SCI论文共有728篇,其中完全由中国学者完成的181篇,与其他国家合作完成的62篇,合计约占全世界的33.38%,但高水平论文数量还有待提高。2017年的研究进展主要集中在油菜籽含油量及品质、油菜籽产量、基因组驯化、雄性不育、非生物胁迫及抗病育种等方面。这些成果将积极地推动油菜育种产业的高产、优质及多元化发展,为我国油菜分子设计育种的实现提供了重要的理论基础。展开更多
The Chinese tree shrew （Tupaia belangeri chinensis） a squirrel-like and rat-sized mammal, has a wide distribution in Southeast Asia, South and Southwest China and has many unique characteristics that make it suitabl...The Chinese tree shrew （Tupaia belangeri chinensis） a squirrel-like and rat-sized mammal, has a wide distribution in Southeast Asia, South and Southwest China and has many unique characteristics that make it suitable for use as an experimental animal. There have been many studies using the tree shrew （Tupaia belangeri） aimed at increasing our understanding of fundamental biological mechanisms and for the modeling of human diseases and therapeutic responses. The recent release of a publicly available annotated genome sequence of the Chinese tree shrew and its genome database （www.treeshrewdb.org） has offered a solid base from which it is possible to elucidate the basic biological properties and create animal models using this species. The extensive characterization of key factors and signaling pathways in the immune and nervous systems has shown that tree shrews possess both conserved and unique features relative to primates. Hitherto, the tree shrew has been successfully used to create animal models for myopia, depression, breast cancer, alcohol-induced or non-alcoholic fatty liver diseases, herpes simplex virus type 1 （HSV-1） and hepatitis C virus （HCV） infections, to name a few. The recent successful genetic manipulation of the tree shrew has opened a new avenue for the wider usage of this animal in biomedical research. In this opinion paper, I attempt to summarize the recent research advances that have used the Chinese tree shrew, with a focus on the new knowledge obtained by using the biological properties identified using the tree shrew genome, a proposal for the genome-based approach for creating animal models, and the genetic manipulation of the tree shrew. With more studies using this species and the application of cutting-edge gene editing techniques, the tree shrew will continue to be under the spot light as a viable animal model for investigating the basis of many different human diseases.展开更多
基金This study was supported by the National Basic Research Program （973 Program） （Nos. 2010CB945401 and 2012CB911201）, the National Natural Science Foundation of China （Grant Nos. 91019020, 81330055, and 31371508）.
文摘Genome editing tools such as the clustered regularly interspaced short palindromic repeat （CRISPR）-associated system （Cas） have been widely used to modify genes in model systems including animal zygotes and human cells, and hold tremendous promise for both basic research and clinical applications. To date, a serious knowledge gap remains in our understanding of DNA repair mechanisms in human early embryos, and in the efficiency and potential off-target effects of using technologies such as CRISPR/Cas9 in human pre-implantation embryos. In this report, we used tripronuclear （3PN） zygotes to further investigate CRISPR/Cas9-mediated gene editing in human cells. We found that CRISPR/Cas9 could effectively cleave the endogenous β-globin gene （HBB）. However, the efficiency of homologous recombination directed repair （HDR） of HBB was low and the edited embryos were mosaic. Off-target cleavage was also apparent in these 3PN zygotes as revealed by the T7E1 assay and whole-exome sequencing. Furthermore, the endogenous delta-globin gene （HBD）, which is homologous to HBB, competed with exogenous donor oligos to act as the repair template, leading to untoward mutations. Our data also indicated that repair of the HBB locus in these embryos occurred preferentially through the non-crossover HDR pathway. Taken together, our work highlights the pressing need to further improve the fidelity and specificity of the CRISPR/Cas9 platform, a prerequisite for any clinical applications of CRSIPR/Cas9-mediated editing.
文摘油菜是我国重要的油料作物,常年种植面积约1亿亩,每年可生产约450万t菜籽油,占国内植物油总消费量的19.7%。与发达国家相比,我国油菜产业主要问题是产量低、品质差,年进口油菜籽约500万t。油菜基因组测序的完成,极大地推动了油菜育种行业的科研工作。据统计(Web of Science检索),2017年与油菜育种相关的SCI论文共有728篇,其中完全由中国学者完成的181篇,与其他国家合作完成的62篇,合计约占全世界的33.38%,但高水平论文数量还有待提高。2017年的研究进展主要集中在油菜籽含油量及品质、油菜籽产量、基因组驯化、雄性不育、非生物胁迫及抗病育种等方面。这些成果将积极地推动油菜育种产业的高产、优质及多元化发展,为我国油菜分子设计育种的实现提供了重要的理论基础。
基金supported by the grant of the National Natural Science Foundation of China(NSFC U1402224)the Chinese Academy of Sciences(CAS zsys-02)
文摘The Chinese tree shrew （Tupaia belangeri chinensis） a squirrel-like and rat-sized mammal, has a wide distribution in Southeast Asia, South and Southwest China and has many unique characteristics that make it suitable for use as an experimental animal. There have been many studies using the tree shrew （Tupaia belangeri） aimed at increasing our understanding of fundamental biological mechanisms and for the modeling of human diseases and therapeutic responses. The recent release of a publicly available annotated genome sequence of the Chinese tree shrew and its genome database （www.treeshrewdb.org） has offered a solid base from which it is possible to elucidate the basic biological properties and create animal models using this species. The extensive characterization of key factors and signaling pathways in the immune and nervous systems has shown that tree shrews possess both conserved and unique features relative to primates. Hitherto, the tree shrew has been successfully used to create animal models for myopia, depression, breast cancer, alcohol-induced or non-alcoholic fatty liver diseases, herpes simplex virus type 1 （HSV-1） and hepatitis C virus （HCV） infections, to name a few. The recent successful genetic manipulation of the tree shrew has opened a new avenue for the wider usage of this animal in biomedical research. In this opinion paper, I attempt to summarize the recent research advances that have used the Chinese tree shrew, with a focus on the new knowledge obtained by using the biological properties identified using the tree shrew genome, a proposal for the genome-based approach for creating animal models, and the genetic manipulation of the tree shrew. With more studies using this species and the application of cutting-edge gene editing techniques, the tree shrew will continue to be under the spot light as a viable animal model for investigating the basis of many different human diseases.
