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In Vivo Exon Replacement in the Mouse Atp7b Gene by the Cas9 System

文献类型：期刊论文

作者Liu, Lili2; Chang, Qiurong2; Xing, Fengying2; Yan, Guofeng2; Fu, Li2; Wang, Huiyang2; Ma, Zhengwen2; Chen, Xuejin2; Li, Yao2; Cao, Jianchang1
刊名HUMAN GENE THERAPY
出版日期2019
卷号30期号:9页码:1079-1092
ISSN号1043-0342
关键词exon replacement CRISPR Cas9 rAAV ATP7B
DOI10.1089/hum.2019.037
文献子类Article
英文摘要The application of CRISPR/Cas9 has opened a new era in gene therapy, making it possible to correct mutated genomes in vivo. Exon replacement can correct many mutations and has potential clinical value. In this study, we used a lentivirus-delivered transgene to obtain transgenic mice in which Cas9 and green fluorescent protein (GFP) were driven by the hTBG promoter and were specifically expressed in the liver. In Cas9-positive mice, only similar to 11.6% of hepatocytes were GFP positive. The newborn Cas9-positive F1 mice were injected via the temporal vein with rAAV carrying a modified homologous replacement sequence for exon 8 of Atp7b and a pair of single-strand guide RNAs targeting the introns surrounding exon 8. When the Cas9-positive hepatocytes were sorted and analyzed by PCR and next-generation deep sequencing with different labels, similar to 16.34 +/- 4.02% to 19.37 +/- 6.50% of the analyzed copies of exon 8 were replaced by the donor template in the genome of GFP-positive hepatocytes, that is, 1.81 +/- 0.29% to 2.09 +/- 0.54% replacement occurred in all liver genomes. However, when rAAV carrying a modified homologous replacement sequence was injected into the adult spCas9 mice, a double-cut deletion ratio of up to 99%, only about 1.10-1.13% of the exon 8 replacement rate was detected in Cas9-positive hepatocytes. This study is the first to achieve exon replacement via CRISPR/Cas9, which will benefit research on CRISPR/Cas9 technology for gene therapy.
学科主题Biotechnology & Applied Microbiology ; Genetics & Heredity ; Research & Experimental Medicine
WOS关键词WILSON-DISEASE MUTATIONS ; BINDING GLOBULIN GENE ; LIVER ; DNA ; EXPRESSION ; EFFICIENT ; THERAPY ; REPEATS ; MODEL ; MICE
语种英语
出版者MARY ANN LIEBERT, INC
WOS记录号WOS:000474172600001
版本出版稿
源URL[http://202.127.25.144/handle/331004/547]  
专题中国科学院上海生命科学研究院营养科学研究所
作者单位1.Univ Chinese Acad Sci, Chinese Acad Sci, CAS Key Lab Tissue Microenvironm & Tumor, Shanghai Inst Nutr & Hlth,Shanghai Inst Biol Sci, Shanghai, Peoples R China;
2.Shanghai Jiao Tong Univ, Sch Med, Dept Lab Anim Sci, Shanghai, Peoples R China;
3.Kunming Univ Sci & Technol, Inst Primate Translat Med, Yunnan Key Lab Primate Biomed Res, Kunming, Yunnan, Peoples R China,
推荐引用方式
GB/T 7714		 Liu, Lili,Chang, Qiurong,Xing, Fengying,et al. In Vivo Exon Replacement in the Mouse Atp7b Gene by the Cas9 System[J]. HUMAN GENE THERAPY,2019,30(9):1079-1092.
APA Liu, Lili.,Chang, Qiurong.,Xing, Fengying.,Yan, Guofeng.,Fu, Li.,...&,.(2019).In Vivo Exon Replacement in the Mouse Atp7b Gene by the Cas9 System.HUMAN GENE THERAPY,30(9),1079-1092.
MLA Liu, Lili,et al."In Vivo Exon Replacement in the Mouse Atp7b Gene by the Cas9 System".HUMAN GENE THERAPY 30.9(2019):1079-1092.

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来源：上海营养与健康研究所

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