公开(公告)号：US20150050699A1

公开(公告)日：2015-02-19

申请号：US14385857

申请日：2013-03-20

申请人： VILNIUS UNIVERSITY

发明人： Virginijus Siksnys ,  Giedrius Gasiunas ,  Tautvydad Karvelis ,  Arvydas Lubys ,  Lolita Zaliauskiene ,  Monika Glemzaite ,  Anja Smith

IPC分类号： C12N15/90 ,  C12N9/22 ,  C12N15/113 ,  C12N15/10

CPC分类号： C12N15/113 ,  C12N9/22 ,  C12N15/102 ,  C12N15/902 ,  C12N15/907 ,  C12N2310/12 ,  C12N2310/14 ,  C12N2310/20 ,  C12N2310/3513 ,  C12N2310/531 ,  C12N2320/00 ,  C12N2800/80 ,  C12P19/34 ,  C12Q1/6811 ,  C12Q2521/301 ,  C12Q2522/101 ,  C12Q2525/121 ,  C12Q2525/151 ,  C12Q2525/301

摘要： Isolation or in vitro assembly of the Cas9-crRNA complex of the Streptococcus thermophilus CRISPR3/Cas system and use for cleavage of DNA bearing a nucleotide sequence complementary to the crRNA and a proto-spacer adjacent motif. Methods for site-specific modification of a target DNA molecule in vitro or in vivo using an RNA-guided DNA endonuclease comprising RNA sequences and at least one of an RuvC active site motif and an HNH active site motif; for conversion of Cas9 polypeptide into a nickase cleaving one strand of double-stranded DNA by inactivating one of the active sites (RuvC or HNH) in the polypeptide by at least one point mutation; for assembly of active polypeptide-polyribonucleotides complex in vivo or in vitro; and for re-programming a Cas9-crRNA complex specificity in vitro and using a cassette containing a single repeat-spacer-repeat unit.

摘要翻译： 分离或体外装配嗜热链球菌CRISPR3 / Cas系统的Cas9-crRNA复合物，并用于裂解含有与crRNA互补的核苷酸序列的DNA和原间隔物相邻基序。 使用包含RNA序列和至少一个RuvC活性位点基序和HNH活性位点基序的RNA引导的DNA核酸内切酶在体外或体内对靶DNA分子进行位点特异性修饰的方法; 用于通过使多肽中的一个活性位点（RuvC或HNH）灭活至少一个点突变，将Cas9多肽转化成切割酶，从而切割一条双链DNA; 用于在体内或体外组装活性多肽 - 多核苷酸复合物; 以及用于在体外重新编程Cas9-crRNA复合物特异性并使用含有单个重复 - 间隔 - 重复单元的盒。