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    • 65. 发明申请
    • GENE EDITING IN THE OOCYTE BY CAS9 NUCLEASES
    • 由CAS9核苷酸在基因组编码
    • WO2014131833A1
    • 2014-09-04
    • PCT/EP2014/053840
    • 2014-02-27
    • HELMHOLTZ ZENTRUM MÜNCHEN DEUTSCHES FORSCHUNGSZENTRUM FÜR GESUNDHEIT UND UMWELT (GMBH)
    • KÜHN, RalfWURST, WolfgangORTIZ SANCHEZ, Oskar
    • A01K67/027C12N9/22C12N15/89C12N15/90
    • C12N5/0609A01K67/0275A01K2217/072A01K2217/075A01K2227/105A01K2267/03A61D19/04C12N9/22C12N15/102C12N15/89C12N15/907C12N2800/80
    • The present invention relates to a method of producing a non-human, mammalian oocyte carrying a modified target sequence in its genome, the method comprising the steps of introducing into a non-human, mammalian oocyte: (a) a clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated protein 9 (Cas9 protein) or a nucleic acid molecule encoding said Cas9 protein; and (b-i) a target sequence specific CRISPR RNA (crRNA) and a trans-activating crRNA (tracr RNA) or a nucleic acid molecule encoding said RNAs; or (b-ii) a chimaeric RNA sequence comprising a target sequence specific crRNA and tracrRNA or a nucleic acid molecule encoding said RNA; wherein the Cas9 protein introduced in (a) and the RNA sequence(s) introduced in (b-i) or (b-ii) form a protein/RNA complex that specifically binds to the target sequence and introduces a single or double strand break within the target sequence. The present invention further relates to the method of the invention, wherein the target sequence is modified by homologous recombination with a donor nucleic acid sequence further comprising the step: (c) introducing a nucleic acid molecule into the cell, wherein the nucleic acid molecule comprises the donor nucleic acid sequence and regions homologous to the target sequence. The present invention also relates to a method of producing a non-human mammal carrying a modified target sequence in its genome.
    • 本发明涉及在其基因组中产生携带修饰的靶序列的非人哺乳动物卵母细胞的方法,所述方法包括以下步骤:向非人哺乳动物卵母细胞中引入:(a)聚集的,规则间隔的, 短回文重复(CRISPR)相关蛋白9(Cas9蛋白)或编码所述Cas9蛋白的核酸分子; 和(b-i)靶序列特异性CRISPR RNA(crRNA)和反式激活性crRNA(tracr RNA)或编码所述RNA的核酸分子; 或(b-ii)包含靶序列特异性crRNA和tracrRNA的嵌合RNA序列或编码所述RNA的核酸分子; 其中在(a)中引入的Cas9蛋白质和引入到(bi)或(b-ii)中的RNA序列形成特异性结合靶序列的蛋白质/ RNA复合物,并引入单链或双链断裂 目标序列。 本发明还涉及本发明的方法,其中通过与供体核酸序列同源重组修饰靶序列,其进一步包括以下步骤:(c)将核酸分子引入细胞,其中所述核酸分子包含 供体核酸序列和与靶序列同源的区域。 本发明还涉及在其基因组中生产携带修饰的靶序列的非人哺乳动物的方法。