This study aimed to compare the efficiencies of clustered regulatory interspaced short palindromic repeat（CRISPR）/Cas9-mediated gene knock-ins with zinc finger nucleases（ZFNs） and transcription activator-like effe...This study aimed to compare the efficiencies of clustered regulatory interspaced short palindromic repeat（CRISPR）/Cas9-mediated gene knock-ins with zinc finger nucleases（ZFNs） and transcription activator-like effector nucleases（TALENs） in bovine and dairy goat fetal fibroblasts. To test the knock-in efficiency, a set of ZFNs and CRISPR/Cas9 plasmids were designed to edit the bovine myostatin（MSTN） gene at exon 2, while a set of TALENs and CRISPR/Cas9 plasmids were designed for editing the dairy goat β-casein gene at exon 2. Donor plasmids utilizing the ZFNs, TALENs, and CRISPR/Cas9 cutting sites were constructed in theGFP-PGK-Neo R plasmid background, including a 5′ and 3′ homologous arm flanking the genes humanized Fat-1（h Fat-1） or enhanced green fluorescent protein（eGFP）. Subsequently, the ZFNs, TALENs, or CRISPR/Cas9 and thehFat-1 or eGFP plasmids were co-transfected by electroporation into bovine and dairy goat fetal fibroblasts. After G418（Geneticin） selection, single cells were obtained by mouth pipetting, flow cytometry or a cell shove. The gene knock-in events were screened by PCR across the homologous arms. The results showed that in bovine fetal fibrobalsts, the efficiencies of ZFNs-mediated eGFP andhFat-1 gene knock-ins were 13.68 and 0%, respectively. The efficiencies of CRISPR/Cas9-mediated eGFP andhFat-1 gene knock-ins were 77.02 and 79.01%, respectively. The eGFP gene knock-in efficiency using CRISPR/Cas9 was about 5.6 times higher than when using the ZFNs gene editing system. Additionally, thehFat-1 gene knock-in was only obtained when using the CRISPR/Cas9 system. The difference of knockin efficiencies between the ZFNs and CRISPR/Cas9 systems were extremely significant（P〈0.01）. In the dairy goat fetal fibroblasts, the efficiencies of TALENs-mediated eGFP andhFat-1 gene knock-ins were 32.35 and 26.47%, respectively. Theefficiencies of eGFP and hFat-1 gene knock-ins using CRISPR/Cas9 were 70.37 and 74.29%, respectively. The knock-in efficiencies difference between the TALENs and CRISPR/Cas9 systems were extremely significant（P〈0.01）. This study demonstrated that CRISPR/Cas9 was more efficient at gene knock-ins in domesticated animal cells than ZFNs and TALENs. The CRISPR/Cas9 technology offers a new era of precise gene editing in domesticated animal cell lines.展开更多
Duchenne Muscular Dystrophy (DMD) is a severe childhood form of muscular dystrophy. Both the severe form and its milder form of Becker Muscular Dystrophy (BMD) are caused by the mutation of dystrophin gene. Different ...Duchenne Muscular Dystrophy (DMD) is a severe childhood form of muscular dystrophy. Both the severe form and its milder form of Becker Muscular Dystrophy (BMD) are caused by the mutation of dystrophin gene. Different from some other genetic diseases such as hemophilia that can be treated by replacement therapy, there is no effective therapy for muscular dystrophy in conventional medication. Gene editing technology from the recently developed engineered nucleases such as TALENs has been successfully employed in genome modification of a variety of species, and will be applied in gene therapy of selected human diseases. The genetic basis of DMD and BMD indicates that DMD is a good target for gene therapy through returning the reading frame of dystrophin gene. Gene therapy strategies described here may apply to many other genetic diseases. Wider application of TALENs in gene therapy have the potential to dramatically prolong the lifespan of individuals with genetic diseases.展开更多
Precise genome modification with engineered nucleases is a powerful tool for studying basic biology and applied biotechnology. Transcription activator-like effector nucleases（TALENs）,consisting of an engineered spec...Precise genome modification with engineered nucleases is a powerful tool for studying basic biology and applied biotechnology. Transcription activator-like effector nucleases（TALENs）,consisting of an engineered specific（TALE） DNA binding domain and a Fok I cleavage domain,are newly developed versatile reagents for genome engineering in different organisms.Because of the simplicity of the DNA recognition code and their modular assembly,TALENs can act as customizable molecular DNA scissors inducing double-strand breaks（DSBs） at given genomic location.Thus,they provide a valuable approach to targeted genome modifications such as mutations, insertions,replacements or chromosome rearrangements.In this article,we review the development of TALENs,and summarize the principles and tools for TALEN-mediated gene targeting in plant cells,as well as current and potential strategies for use in plant research and crop improvement.展开更多
Mitochondrial diseases are maternally inherited hetero- geneous disorders that are primarily caused by mitochondrial DNA （mtDNA） mutations. Depending on the ratio of mutant to wild-type mtDNA, known as heteroplasmy,...Mitochondrial diseases are maternally inherited hetero- geneous disorders that are primarily caused by mitochondrial DNA （mtDNA） mutations. Depending on the ratio of mutant to wild-type mtDNA, known as heteroplasmy, mitochondrial defects can result in a wide spectrum of clinical manifestations. Mitochondria-targeted endonucleases provide an alternative avenue for treating mitochondrial disorders via targeted destruc- tion of the mutant mtDNA and induction of heteroplasmic shifting. Here, we generated mitochondrial disease patient-specific induced pluripotent stem cells （MiPSCs） that harbored a high proportion of m.3243A〉G mtDNA mutations and caused mitochondrial encephalomyopathy and stroke-like episodes （MELAS）. We engineered mitochondrial-targeted transcription activator-like effector nucleases （mitoTALENs） and successfully eliminated the m.3243A〉G mutation in MiPSCs. Off-target mutagenesis was not detected in the targeted MiPSC clones. Utilizing a dual fluorescence iPSC reporter cell line expressing a 3243G mutant mtDNA sequence in the nuclear genome, mitoTALENs displayed a significantly limited ability to target the nuclear genome compared with nuclear-localized TALENs. Moreover, genetically rescued MiPSCs displayed normal mitochondrial respiration and energy production. Moreover, neuronal progenitor cells differentiated from the rescued MiPSCs also demonstrated normal metabolic profiles. Further- more, we successfully achieved reduction in the human m.3243A〉G mtDNA mutation in porcine oocytes via injection of mitoTALEN mRNA. Our study shows the great potential for using mitoTALENs for specific targeting of mutant mtDNA both in iPSCs and mammalian oocytes, which not only provides a new avenue for studying mitochondrial biology and disease but also suggests a potential therapeutic approach for the treatment of mitochondrial disease, as well as the prevention of germline transmission of mutant mtDNA.展开更多
Background:Zinc-finger nucleases(ZFNs)and transcription activator-like effector nucleases(TALENs)have been successfully used to knock out endogenous genes in stem cell research.However,the deficiencies of current gene...Background:Zinc-finger nucleases(ZFNs)and transcription activator-like effector nucleases(TALENs)have been successfully used to knock out endogenous genes in stem cell research.However,the deficiencies of current gene-based delivery systems may hamper the clinical application of these nucleases.A new delivery method that can improve the utility of these nucleases is needed.Results:In this study,we utilized a cell-penetrating peptide-based system for ZFN and TALEN delivery.Functional TAT-ZFN and TAT-TALEN proteins were generated by fusing the cell-penetrating TAT peptide to ZFN and TALEN,respectively.However,TAT-ZFN was difficult to purify in quantities sufficient for analysis in cell culture.Purified TAT-TALEN was able to penetrate cells and disrupt the gene encoding endogenous human chemokine(C-C motif)receptor 5(CCR5,a co-receptor for HIV-1 entry into cells).Hypothermic treatment greatly enhanced the TAT-TALENmediated gene disruption efficiency.A 5%modification rate was observed in human induced pluripotent stem cells(hiPSCs)treated with TAT-TALEN as measured by the Surveyor assay.Conclusions:TAT-TALEN protein-mediated gene disruption was applicable in hiPSCs and represents a promising technique for gene knockout in stem cells.This new technique may advance the clinical application of TALEN technology.展开更多
基金supported by the National Transgenic Project of China (2016ZX08010001-002)the National Natural Science Foundation of China (81471001)+1 种基金the Inner Mongolia Science and Technology Program, China (201502073)the National 863 Prgram of China (2009AA10Z111)
文摘This study aimed to compare the efficiencies of clustered regulatory interspaced short palindromic repeat（CRISPR）/Cas9-mediated gene knock-ins with zinc finger nucleases（ZFNs） and transcription activator-like effector nucleases（TALENs） in bovine and dairy goat fetal fibroblasts. To test the knock-in efficiency, a set of ZFNs and CRISPR/Cas9 plasmids were designed to edit the bovine myostatin（MSTN） gene at exon 2, while a set of TALENs and CRISPR/Cas9 plasmids were designed for editing the dairy goat β-casein gene at exon 2. Donor plasmids utilizing the ZFNs, TALENs, and CRISPR/Cas9 cutting sites were constructed in theGFP-PGK-Neo R plasmid background, including a 5′ and 3′ homologous arm flanking the genes humanized Fat-1（h Fat-1） or enhanced green fluorescent protein（eGFP）. Subsequently, the ZFNs, TALENs, or CRISPR/Cas9 and thehFat-1 or eGFP plasmids were co-transfected by electroporation into bovine and dairy goat fetal fibroblasts. After G418（Geneticin） selection, single cells were obtained by mouth pipetting, flow cytometry or a cell shove. The gene knock-in events were screened by PCR across the homologous arms. The results showed that in bovine fetal fibrobalsts, the efficiencies of ZFNs-mediated eGFP andhFat-1 gene knock-ins were 13.68 and 0%, respectively. The efficiencies of CRISPR/Cas9-mediated eGFP andhFat-1 gene knock-ins were 77.02 and 79.01%, respectively. The eGFP gene knock-in efficiency using CRISPR/Cas9 was about 5.6 times higher than when using the ZFNs gene editing system. Additionally, thehFat-1 gene knock-in was only obtained when using the CRISPR/Cas9 system. The difference of knockin efficiencies between the ZFNs and CRISPR/Cas9 systems were extremely significant（P〈0.01）. In the dairy goat fetal fibroblasts, the efficiencies of TALENs-mediated eGFP andhFat-1 gene knock-ins were 32.35 and 26.47%, respectively. Theefficiencies of eGFP and hFat-1 gene knock-ins using CRISPR/Cas9 were 70.37 and 74.29%, respectively. The knock-in efficiencies difference between the TALENs and CRISPR/Cas9 systems were extremely significant（P〈0.01）. This study demonstrated that CRISPR/Cas9 was more efficient at gene knock-ins in domesticated animal cells than ZFNs and TALENs. The CRISPR/Cas9 technology offers a new era of precise gene editing in domesticated animal cell lines.
文摘Duchenne Muscular Dystrophy (DMD) is a severe childhood form of muscular dystrophy. Both the severe form and its milder form of Becker Muscular Dystrophy (BMD) are caused by the mutation of dystrophin gene. Different from some other genetic diseases such as hemophilia that can be treated by replacement therapy, there is no effective therapy for muscular dystrophy in conventional medication. Gene editing technology from the recently developed engineered nucleases such as TALENs has been successfully employed in genome modification of a variety of species, and will be applied in gene therapy of selected human diseases. The genetic basis of DMD and BMD indicates that DMD is a good target for gene therapy through returning the reading frame of dystrophin gene. Gene therapy strategies described here may apply to many other genetic diseases. Wider application of TALENs in gene therapy have the potential to dramatically prolong the lifespan of individuals with genetic diseases.
基金supported by the National Natural Science Foundation of China(Grant Nos.201263,383601 and 31200273)
文摘Precise genome modification with engineered nucleases is a powerful tool for studying basic biology and applied biotechnology. Transcription activator-like effector nucleases（TALENs）,consisting of an engineered specific（TALE） DNA binding domain and a Fok I cleavage domain,are newly developed versatile reagents for genome engineering in different organisms.Because of the simplicity of the DNA recognition code and their modular assembly,TALENs can act as customizable molecular DNA scissors inducing double-strand breaks（DSBs） at given genomic location.Thus,they provide a valuable approach to targeted genome modifications such as mutations, insertions,replacements or chromosome rearrangements.In this article,we review the development of TALENs,and summarize the principles and tools for TALEN-mediated gene targeting in plant cells,as well as current and potential strategies for use in plant research and crop improvement.
基金This work was supported in part by the ＂Reproductive health and major birth defects prevention and control research＂ Key Special Fund （No. 2016YFC1000601）, the National Natural Science Foundation of China （Grant Nos. 31371521, 81370766, 81401254, 81570101, 81671121, 31601187, 81521002）, the Guangdong Province Science and Technology Project （2014TQ01R683, 2017A020 214005, 2016A020216023, 2015A030310119, 2016B030229008）, the Bureau of Science and Technology of Guangzhou Municipality （201505011111498）, the ＂Reproductive health and major birth defects prevention and control research＂ Key Special Fund （Nos. 2016YFC1000201 and 2016YFC1000302）, the Ministry of Science and Technology of China Grants （973 program 2014CB943203）, and the Beijing Nova Program （xxjh2015011）.
文摘Mitochondrial diseases are maternally inherited hetero- geneous disorders that are primarily caused by mitochondrial DNA （mtDNA） mutations. Depending on the ratio of mutant to wild-type mtDNA, known as heteroplasmy, mitochondrial defects can result in a wide spectrum of clinical manifestations. Mitochondria-targeted endonucleases provide an alternative avenue for treating mitochondrial disorders via targeted destruc- tion of the mutant mtDNA and induction of heteroplasmic shifting. Here, we generated mitochondrial disease patient-specific induced pluripotent stem cells （MiPSCs） that harbored a high proportion of m.3243A〉G mtDNA mutations and caused mitochondrial encephalomyopathy and stroke-like episodes （MELAS）. We engineered mitochondrial-targeted transcription activator-like effector nucleases （mitoTALENs） and successfully eliminated the m.3243A〉G mutation in MiPSCs. Off-target mutagenesis was not detected in the targeted MiPSC clones. Utilizing a dual fluorescence iPSC reporter cell line expressing a 3243G mutant mtDNA sequence in the nuclear genome, mitoTALENs displayed a significantly limited ability to target the nuclear genome compared with nuclear-localized TALENs. Moreover, genetically rescued MiPSCs displayed normal mitochondrial respiration and energy production. Moreover, neuronal progenitor cells differentiated from the rescued MiPSCs also demonstrated normal metabolic profiles. Further- more, we successfully achieved reduction in the human m.3243A〉G mtDNA mutation in porcine oocytes via injection of mitoTALEN mRNA. Our study shows the great potential for using mitoTALENs for specific targeting of mutant mtDNA both in iPSCs and mammalian oocytes, which not only provides a new avenue for studying mitochondrial biology and disease but also suggests a potential therapeutic approach for the treatment of mitochondrial disease, as well as the prevention of germline transmission of mutant mtDNA.
基金We are grateful to Miguel A.Esteban and his group for supplying the hiPSCs.This work was financially supported by the National Science and Technology Major Project(2013ZX10001-004-002-004)the National Natural Science Foundation of China(No.81200398).
文摘Background:Zinc-finger nucleases(ZFNs)and transcription activator-like effector nucleases(TALENs)have been successfully used to knock out endogenous genes in stem cell research.However,the deficiencies of current gene-based delivery systems may hamper the clinical application of these nucleases.A new delivery method that can improve the utility of these nucleases is needed.Results:In this study,we utilized a cell-penetrating peptide-based system for ZFN and TALEN delivery.Functional TAT-ZFN and TAT-TALEN proteins were generated by fusing the cell-penetrating TAT peptide to ZFN and TALEN,respectively.However,TAT-ZFN was difficult to purify in quantities sufficient for analysis in cell culture.Purified TAT-TALEN was able to penetrate cells and disrupt the gene encoding endogenous human chemokine(C-C motif)receptor 5(CCR5,a co-receptor for HIV-1 entry into cells).Hypothermic treatment greatly enhanced the TAT-TALENmediated gene disruption efficiency.A 5%modification rate was observed in human induced pluripotent stem cells(hiPSCs)treated with TAT-TALEN as measured by the Surveyor assay.Conclusions:TAT-TALEN protein-mediated gene disruption was applicable in hiPSCs and represents a promising technique for gene knockout in stem cells.This new technique may advance the clinical application of TALEN technology.
