Gene editing has recently emerged as a promising technology to engineer genetic modifications precisely in the genome to achieve long-term relief from corneal disorders.Recent advances in the molecular biology leading...Gene editing has recently emerged as a promising technology to engineer genetic modifications precisely in the genome to achieve long-term relief from corneal disorders.Recent advances in the molecular biology leading to the development of clustered regularly interspaced short palindromic repeats(CRISPRs) and CRISPR-associated systems,zinc finger nucleases and transcription activator like effector nucleases have ushered in a new era for high throughput in vitro and in vivo genome engineering.Genome editing can be successfully used to decipher complex molecular mechanisms underlying disease pathophysiology,develop innovative next generation gene therapy,stem cell-based regenerative therapy,and personalized medicine for corneal and other ocular diseases.In this review we describe latest developments in the field of genome editing,current challenges,and future prospects for the development of personalized genebased medicine for corneal diseases.The gene editing approach is expected to revolutionize current diagnostic and treatment practices for curing blindness.展开更多
Recently, an epoch-making genome engineering technology using clustered regularly at interspaced short palindromic repeats(CRISPR) and CRISPR associated(Cas) nucleases, was developed. Previous technologies for genome ...Recently, an epoch-making genome engineering technology using clustered regularly at interspaced short palindromic repeats(CRISPR) and CRISPR associated(Cas) nucleases, was developed. Previous technologies for genome manipulation require the time-consuming design and construction of genome-engineered nucleases for each target and have, therefore, not been widely used in mouse research where standard techniques based on homologous recombination are commonly used. The CRISPR/Cas system only requires the design of sequences complementary to a target locus, making this technology fast and straightforward. In addition, CRISPR/Cas can be used to generate mice carrying mutations in multiple genes in a single step, an achievement not possible using other methods. Here, we review the uses of this technology in genetic analysis and manipulation, including achievements made possible to date and the prospects for future therapeutic applications.展开更多
Genome editing using the Cas9 endonuclease of Streptococcus pyogenes has demonstrated unparalleled efficacy and facility for modifying genomes in a wide variety of organisms. Caenorhabditis elegans is one of the most ...Genome editing using the Cas9 endonuclease of Streptococcus pyogenes has demonstrated unparalleled efficacy and facility for modifying genomes in a wide variety of organisms. Caenorhabditis elegans is one of the most convenient multicellular organisms for genetic analysis, and the application of this novel genome editing technique to this organism promises to revolutionize analysis of gene function in the future. CRISPR-Cas9 has been successfully used to generate imprecise insertions and deletions via non-homologous end-joining mechanisms and to create precise mutations by homology-directed repair from donor templates. Key variables are the methods used to deliver the Cas9 endonuclease and the efficiency of the single guide RNAs. CRISPR-Cas9-mediated editing appears to be highly specific in C. elegans, with no reported off-target effects. In this review, 1 briefly summarize recent progress in CRISPR-Cas9-based genome editing in C. elegans, highlighting technical improvements in mutagenesis and mutation detection, and discuss potential future appli- cations of this technique.展开更多
文摘Gene editing has recently emerged as a promising technology to engineer genetic modifications precisely in the genome to achieve long-term relief from corneal disorders.Recent advances in the molecular biology leading to the development of clustered regularly interspaced short palindromic repeats(CRISPRs) and CRISPR-associated systems,zinc finger nucleases and transcription activator like effector nucleases have ushered in a new era for high throughput in vitro and in vivo genome engineering.Genome editing can be successfully used to decipher complex molecular mechanisms underlying disease pathophysiology,develop innovative next generation gene therapy,stem cell-based regenerative therapy,and personalized medicine for corneal and other ocular diseases.In this review we describe latest developments in the field of genome editing,current challenges,and future prospects for the development of personalized genebased medicine for corneal diseases.The gene editing approach is expected to revolutionize current diagnostic and treatment practices for curing blindness.
基金Supported by The Grants from the Ministry of EducationCulture+7 种基金SportsScience and Technology of Japanthe Ministry of HealthLabour and Welfare of Japanthe National Institute of Biomedical Innovationthe Asahi Glass Foundationthe Ichiro Kanehara Foundationthe Program for Cultivating Global Leaders in Heavy Ion Therapeutics and Engineering
文摘Recently, an epoch-making genome engineering technology using clustered regularly at interspaced short palindromic repeats(CRISPR) and CRISPR associated(Cas) nucleases, was developed. Previous technologies for genome manipulation require the time-consuming design and construction of genome-engineered nucleases for each target and have, therefore, not been widely used in mouse research where standard techniques based on homologous recombination are commonly used. The CRISPR/Cas system only requires the design of sequences complementary to a target locus, making this technology fast and straightforward. In addition, CRISPR/Cas can be used to generate mice carrying mutations in multiple genes in a single step, an achievement not possible using other methods. Here, we review the uses of this technology in genetic analysis and manipulation, including achievements made possible to date and the prospects for future therapeutic applications.
基金supported by National Institutes of Health (NIH grant R01 GM054657) to A.D.C
文摘Genome editing using the Cas9 endonuclease of Streptococcus pyogenes has demonstrated unparalleled efficacy and facility for modifying genomes in a wide variety of organisms. Caenorhabditis elegans is one of the most convenient multicellular organisms for genetic analysis, and the application of this novel genome editing technique to this organism promises to revolutionize analysis of gene function in the future. CRISPR-Cas9 has been successfully used to generate imprecise insertions and deletions via non-homologous end-joining mechanisms and to create precise mutations by homology-directed repair from donor templates. Key variables are the methods used to deliver the Cas9 endonuclease and the efficiency of the single guide RNAs. CRISPR-Cas9-mediated editing appears to be highly specific in C. elegans, with no reported off-target effects. In this review, 1 briefly summarize recent progress in CRISPR-Cas9-based genome editing in C. elegans, highlighting technical improvements in mutagenesis and mutation detection, and discuss potential future appli- cations of this technique.
基金supported by the National Natural Science Foundation of China(Grant No.31301064,81372453)Natural Science Foundation of Shanghai(Grant No.13ZR1446300)Shanghai Municipal Science and Technology Commission Fund(Grant No.15431902800)~~
文摘该研究利用CRISPR/Cas9(clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9)基因编辑系统构建了TP53(tumor antigen p53)基因敲除He La细胞系。CRISPR/Cas9系统能够精确地切开TP53基因并在双链断裂处插入选择标记(通过与供体质粒进行同源重组获得)。进一步的功能试验表明,TP53基因敲除的He La细胞拥有更强的细胞增殖能力、化疗耐药性以及氧化应激能力,提示He La(TP53–/–)恶性程度增强。所有的数据旨在描述一个简单和有效的方法,即通过CRISPR/Cas9系统来构建基因缺失细胞系,期望在较大程度上帮助研究和阐明基因功能以及细胞机制。
