HELMHOLTZ ZENTRUM MÜNCHEN DEUTSCHES FORSCHUNGSZENTRUM FÜR GESUNDHEIT UND UMWELT (GMBH)
发明人:
KÜHN, Ralf,WURST, Wolfgang,ORTIZ SANCHEZ, Oskar
申请号：
EPEP2014/053840
公开号：
WO2014/131833A1
申请日:
2014.02.27
申请国别(地区):
EP
年份:
2014
代理人:
摘要：
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.La présente invention concerne une méthode de production d'un ovocyte mammifère non humain portant une séquence cible modifiée dans son génome, ladite méthode comprenant les étapes consistant à introduire dans un ovocyte mammifère non humain : (a) une protéine 9 associée à des séquences à séries de répétitions palindromiques courtes régulièrement espacées (CRISPR) (protéine Cas9) ou une molécule d'acide nucléique codant pour ladite protéine Cas9 ； et (b-i) un ARN CRISPR (ARNcr) et un ARNcr de transactivation (ARNtracr) spécifiques à une séquence cible ou une molécule d'ac
