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1. 发明申请

WO2010140066A2 METHOD OF ALTERING NUCLEIC ACIDS 审中-公开
标题翻译：改变核酸的方法
公开(公告)号：WO2010140066A2
公开(公告)日：2010-12-09
申请号：PCT/IB2010/002396
申请日：2010-06-04
申请人： GENE BRIDGES GMBH , STEWART, Adrian, Francis , IACONE, Roberto , MARESCA, Marcello , ANASTASSIADIS, Konstantinos
发明人： STEWART, Adrian, Francis , IACONE, Roberto , MARESCA, Marcello , ANASTASSIADIS, Konstantinos
CPC分类号： C12N15/102 , C12N15/111 , C12N15/113 , C12N15/90 , C12N2310/124 , C12N2320/50
摘要： The invention provides a method for altering the sequence of a target nucleic acid molecule to incorporate a replacement nucleic acid sequence. The method comprising the steps of: a) introducing a nucleic acid fragment into the target nucleic acid molecule by homologous recombination, wherein the nucleic acid fragment comprises: i) a first region of homology to the target nucleic acid molecule; ii) a 5' splice site; iii) a selectable marker; iv) a 3' splice site; v) a second region of homology to the target nucleic acid molecule; wherein components i) to v) are ordered from 5' to 3'; and the replacement nucleic acid sequence is positioned within or between the first and second regions of homology but external of the splice sites; b) selecting for the introduction of the nucleic acid fragment using the selectable marker; c) incubating the product of step b) under conditions suitable for a splicing reaction to occur such that the selectable marker is excised and from the target nucleic acid, and thereby generating the desired altered target nucleic acid sequence.
摘要翻译：本发明提供了改变靶核酸分子序列以掺入置换核酸序列的方法。该方法包括以下步骤：a）通过同源重组将核酸片段引入靶核酸分子，其中所述核酸片段包含：i）与靶核酸分子同源的第一区域; ii）5'剪接位点; iii）可选标记; iv）3'剪接位点; v）与靶核酸分子同源的第二区域; 其中组分i）至v）从5'到3'被排序; 并且所述置换的核酸序列位于所述第一和第二同源区之内或之间，但位于所述剪接位点的外部; b）使用选择性标记选择引入核酸片段; c）在适合于剪接反应的条件下孵育步骤b）的产物，使得切除选择性标记并从靶核酸中产生所需的改变的靶核酸序列。

2. 发明申请

WO2009104094A2 METHOD OF NUCLEIC ACID RECOMBINATION 审中-公开
标题翻译：核酸重组方法
公开(公告)号：WO2009104094A2
公开(公告)日：2009-08-27
申请号：PCT/IB2009/000488
申请日：2009-02-20
申请人： GENE BRIDGES GMBH , MARESCA, Marcello , ERLER, Axel, Steffen , FU, Jun , SEIBERT, Philipp, Martin , STEWART, Adrian, Francis , ZHANG, Youming
发明人： MARESCA, Marcello , ERLER, Axel, Steffen , FU, Jun , SEIBERT, Philipp, Martin , STEWART, Adrian, Francis , ZHANG, Youming
IPC分类号： C12N15/09
CPC分类号： C12N15/1082
摘要： The invention provides a method for inserting a single stranded replacement nucleic acid into a target nucleic acid, said method comprising the steps of: a) generating a single stranded replacement nucleic acid from a double stranded nucleic acid, wherein the double stranded nucleic acid is adapted at one or both of its 5' ends such that preferential degradation of one strand and/or strand separation generates the single stranded replacement nucleic acid, wherein the single stranded replacement nucleic acid comprises a 5' region that is identical to sequence on the target nucleic acid, a 3' region that is identical to sequence on the target nucleic acid and optionally a replacement region between the 5' and 3' regions that is not identical to sequence on the target nucleic acid, b) exposing the target nucleic acid to the single stranded replacement nucleic acid under conditions suitable for recombination to occur between the single stranded replacement nucleic acid and the target nucleic acid, and c) selecting a target nucleic acid whose sequence has been altered by inclusion of said single stranded replacement nucleic acid. Other methods for modifying target nucleic acids are also provided.
摘要翻译：本发明提供了将单链置换核酸插入靶核酸的方法，所述方法包括以下步骤：a）从双链核酸产生单链置换核酸，其中双链核酸适应在其5'末端的一个或两个上，使得一条链和/或链分离的优先降解产生单链置换核酸，其中单链置换核酸包含与靶核酸上的序列相同的5'区酸，3'区，其与靶核酸上的序列相同，并且任选地在与靶核酸上的序列不相同的5'和3'区之间的置换区，b）将靶核酸暴露于单链置换核酸在适于在单链置换核酸和靶核之间发生重组的条件下进行并且c）选择通过包含所述单链置换核酸改变其序列的靶核酸。还提供了用于修饰靶核酸的其它方法。

3. 发明申请

WO2010113031A3 METHOD OF ALTERING NUCLEIC ACIDS 审中-公开
公开(公告)号：WO2010113031A3
公开(公告)日：2010-10-07
申请号：PCT/IB2010/000893
申请日：2010-03-30
申请人： GENE BRIDGES GMBH , STEWART, Adrian, Francis , ZHANG, Youming , MARESCA, Marcello , KRANZ, Harald , NOLL, Stephan
发明人： STEWART, Adrian, Francis , ZHANG, Youming , MARESCA, Marcello , KRANZ, Harald , NOLL, Stephan
IPC分类号： C12N15/10 , C12N15/90
摘要： The invention provides a method for altering the sequence of a target nucleic acid molecule, said method comprising the steps of: a) introducing a nucleic acid fragment into the target nucleic acid molecule by homologous recombination, wherein the nucleic acid fragment comprises: i) a first region of homology to the target nucleic acid molecule; ii) a first recognition sequence for a Type IIS nuclease; iii) a selectable marker; iv) a second recognition sequence for a Type IIS nuclease; v) a second region of homology to the target nucleic acid molecule; wherein components i) to v) are ordered from 5' to 3'; and a replacement nucleic acid sequence positioned between the first and second regions of homology but flanking the recognition sequences for the Type IIS nucleases; b) selecting for the incorporation of the replacement nucleic acid using the selectable marker, and c) cleaving the product of step b) with Type IIS nucleases such that the selectable marker is excised, to produce a linear target fragment including the desired replacement sequence.

4. 发明申请

WO2014033644A2 METHODS OF NUCLEASE-BASED GENETIC ENGINEERING 审中-公开
标题翻译：基于核酶的遗传工程方法
公开(公告)号：WO2014033644A2
公开(公告)日：2014-03-06
申请号：PCT/IB2013/058083
申请日：2013-08-28
申请人： NOVARTIS AG , YANG, Yi , MARESCA, Marcello
发明人： YANG, Yi , MARESCA, Marcello
IPC分类号： C12N15/09
CPC分类号： C12N15/90 , A01K2217/00 , C12N15/102 , C12N15/85 , C12N2800/80
摘要： The present invention relates to genetic techniques employing the direct Iigatation of an external DNA fragment generated in situ by the same ZFNs that target the genome. ObLiGaRe, i.e., the obligated ligation-gated recombination, is a new method for genetic engineering using custom designed nucleases, and a strategy of site-specific gene insertion utilizing the NHEJ pathway. It applies a similar logic to the one used in unidirectional loxP sites(Oberdoerffer et al., 2003) but maintains all the advantages and flexibility of CDNs.
摘要翻译：本发明涉及采用由靶向基因组的相同ZFN原位产生的外部DNA片段的直接连接的遗传技术。 ObliGaRe，即专性连接门控重组，是使用定制设计的核酸酶的基因工程的新方法，以及利用NHEJ途径的位点特异性基因插入策略。它应用了与单向loxP站点类似的逻辑（Oberdoerffer et al。，2003），但保留了CDN的所有优点和灵活性。

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