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    • 3. 发明申请
    • METHODS AND COMPOSITIONS FOR T-RNA BASED GUIDE RNA EXPRESSION
    • 基于T-RNA的指导RNA表达的方法和组合物
    • WO2017105991A1
    • 2017-06-22
    • PCT/US2016/065537
    • 2016-12-08
    • DANISCO US INC.
    • BENDEZU, Felipe OseasFAN, XiaochunFRISCH, Ryan L.HONG, Seung-Pyo
    • C12N15/11C12N15/113C12N15/90C12N15/09C12N15/81
    • C12N15/113C12N15/111C12N15/815C12N2310/20C12N2310/3519C12N2330/51
    • Compositions and methods are provided for editing nucleotides and/or altering target sites in the genome of a cell. The methods and compositions employ a recombinant DNA construct comprising a tRNA promoter operably linked to a polynucleotide encoding a single guide RNA, wherein said recombinant DNA construct does not comprise a nucleotide sequence encoding a ribozyme, wherein said guide RNA is capable of forming a guide RNA/Cas endonuclease complex, wherein said complex can bind to and cleave a target site sequence in the genome of a cell such as a microbial cell. The present disclosure further describes methods and compositions employing a recombinant DNA construct comprising a tRNA promoter operably linked to a spacer sequence and a polynucleotide encoding a single guide RNA, wherein said recombinant DNA construct does not comprise a nucleotide sequence encoding a ribozyme, wherein said guide RNA is capable of forming a guide RNA/Cas endonuclease complex, wherein said complex can bind to and cleave a target site sequence in the genome of a non-conventional yeast.
    • 提供用于编辑核苷酸和/或改变细胞基因组中的靶位点的组合物和方法。 所述方法和组合物采用重组DNA构建体,所述重组DNA构建体包含可操作地连接至编码单个引导RNA的多核苷酸的tRNA启动子,其中所述重组DNA构建体不包含编码核酶的核苷酸序列,其中所述引导RNA能够形成指导RNA / Cas核酸内切酶复合物,其中所述复合物可以结合并切割细胞基因组中的靶位点序列,例如微生物细胞。 本公开进一步描述了采用重组DNA构建体的方法和组合物,所述重组DNA构建体包含可操作地连接至间隔序列的tRNA启动子和编码单个引导RNA的多核苷酸,其中所述重组DNA构建体不包含编码核酶的核苷酸序列, RNA能够形成引导RNA / Cas核酸内切酶复合物,其中所述复合物可以结合并切割非常规酵母的基因组中的靶位点序列。