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
Objective: To explore the possibility of using traditional Chinese medicine (TCM) in treating β thalassemia, its clinical effect and molecular mechanism of the action.Methods: According to the TCM theory of“Shen pro...Objective: To explore the possibility of using traditional Chinese medicine (TCM) in treating β thalassemia, its clinical effect and molecular mechanism of the action.Methods: According to the TCM theory of“Shen producing marrow”, the composite recipe, Yisui Shenxueling Granule (YSSXL), consisting of Chinese drugs for nourishing Shen and supplementing marrow (NS&SM) was given orally to 7 8 patients with β thalassemia (49 of the severe type and 29 of moderate type ), 3 times a day, 10 g each time (for children, the dose would be reduced proper ly), with 3 months as one therapeutic course, and no blood transfusion used in t he course. The clinical therapeutic efficacy and hematologic parameters in patie nts were observed, and systemic gene analysis was conducted with PAGE, PCR, PCR SSCP, RT PCR and DNA sequences analysis and mRNA detection, in order to s tudy the molecular mechanism from the relationships between genetic mutation and clinical efficacy, gene expression and its regulation. Results: YSSXL showed obvious therapeutic effect in treating β thalassemia. Gene analysis revealed that it did not change the genetic mutatio n type, but could obviously increase hemoglobin, fetal hemoglobin (HbF), γ/(β+ γ) globin ratio, γ globin mRNA expression and GM CSF mRNA expression in patients, as well as the GM CSFmRMA in marrow of mice after 60 Co radia tion. Conclusion: YSSXL has a remarkable therapeutic effect on β tha lassemia, and its possible mechanism is its action in unlocking γ gene, in creasing the γ globin expression and enhancing HbF synthesis so as to compe nsate for the gene defect. This study has opened a new path for the treatment of β thalassemia with TCM.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.
基金This item was supported by National Funds of Natural Sciences (No. 3017119 9) and Natural Science Foundation of Guangxi Autonomous Region (No. 014402C)
文摘Objective: To explore the possibility of using traditional Chinese medicine (TCM) in treating β thalassemia, its clinical effect and molecular mechanism of the action.Methods: According to the TCM theory of“Shen producing marrow”, the composite recipe, Yisui Shenxueling Granule (YSSXL), consisting of Chinese drugs for nourishing Shen and supplementing marrow (NS&SM) was given orally to 7 8 patients with β thalassemia (49 of the severe type and 29 of moderate type ), 3 times a day, 10 g each time (for children, the dose would be reduced proper ly), with 3 months as one therapeutic course, and no blood transfusion used in t he course. The clinical therapeutic efficacy and hematologic parameters in patie nts were observed, and systemic gene analysis was conducted with PAGE, PCR, PCR SSCP, RT PCR and DNA sequences analysis and mRNA detection, in order to s tudy the molecular mechanism from the relationships between genetic mutation and clinical efficacy, gene expression and its regulation. Results: YSSXL showed obvious therapeutic effect in treating β thalassemia. Gene analysis revealed that it did not change the genetic mutatio n type, but could obviously increase hemoglobin, fetal hemoglobin (HbF), γ/(β+ γ) globin ratio, γ globin mRNA expression and GM CSF mRNA expression in patients, as well as the GM CSFmRMA in marrow of mice after 60 Co radia tion. Conclusion: YSSXL has a remarkable therapeutic effect on β tha lassemia, and its possible mechanism is its action in unlocking γ gene, in creasing the γ globin expression and enhancing HbF synthesis so as to compe nsate for the gene defect. This study has opened a new path for the treatment of β thalassemia with TCM.
