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    • 2. 发明申请
    • METHODS AND COMPOSITIONS FOR ENHANCED NUCLEASE-MEDIATED GENOME MODIFICATION AND REDUCED OFF-TARGET SITE EFFECTS
    • 用于增强的核酸酶介导的基因组修饰和减少的非靶标位点效应的方法和组合物
    • WO2017100158A1
    • 2017-06-15
    • PCT/US2016/065070
    • 2016-12-06
    • DANISCO US INC.
    • FRISCH, Ryan L.
    • C12N15/63C12N15/90
    • C12N15/902C12N15/102C12N15/905C12Q2521/301
    • Compositions and methods are provided for editing nucleotides or altering target sites in the genome of a cell. The methods and compositions employ a guide RNA/Cas endonuclease system and at least one component selected from the group consisting of (i) an inhibitor of end joining (NHEJ), (ii) an activator of homology-directed repair (HDR) or (iii) any one combination of (i) and (ii), to provide an effective system for editing nucleotides or altering target sites within the genome of a cell. The present disclosure also describes methods for editing a nucleotide sequence in the genome of a microbial cell employing a guide RNA/Cas endonuclease system and at least one component selected from the group consisting of (i) an inhibitor of NHEJ, (ii) an activator of HDR, or (iii) any one combination of (i) and (ii), wherein said microbial cell has reduced or no off-target site effects.
    • 提供了用于编辑核苷酸或改变细胞基因组中的靶位点的组合物和方法。 所述方法和组合物使用引导RNA / Cas核酸内切酶系统和至少一种选自以下的组分:(i)末端连接抑制剂(NHEJ),(ii)同源性定向修复活化剂(HDR)或( iii)(i)和(ii)的任何一种组合,以提供编辑核苷酸或改变细胞基因组内靶位点的有效系统。 本公开还描述了使用引导RNA / Cas核酸内切酶系统和选自以下的至少一种成分编辑微生物细胞基因组中的核苷酸序列的方法:(i)NHEJ抑制剂,(ii)活化剂 或(iii)(i)和(ii)的任何一种组合,其中所述微生物细胞具有降低的或不具有脱靶部位效应。
    • 7. 发明申请
    • YEAST EXPRESSION VECTOR FOR YEAST CHROMOSOMAL INTEGRATION OR TRANSFORMATION OF INDUSTRIAL STRAINS, AND USE THEREOF
    • YEAST表达载体用于YEAST染色体整合或转化工业菌株及其用途
    • WO2013100713A3
    • 2013-09-19
    • PCT/KR2012011772
    • 2012-12-28
    • UNIV EWHA IND COLLABORATION
    • CHOI WON JAKIM WAN KEE
    • C12N15/81C12N1/19C12N15/65
    • C12N15/81C12N15/905C12N2800/30
    • The present invention relates to a novel dual module expression vector for yeast transformation, and a transformation method of a target nucleotide sequence using the same. An expression module of the present invention comprises: an expression module comprising a multiple cloning site (MCS), a proper promoter, and a terminator; and a rescue module. The vector of the present invention can stably express a target gene in a yeast, particularly, Kluyveromyces marxianus or Saccharomyces cerevisiae, and can integrate a target gene to a specific position in a chromosome with high efficiency. A transformation method using the vector of the present invention can be very usefully applied to industrial production of desired products (biomass) since it is possible to prepare a transgenic yeast strain in which a target gene is stably integrated in a chromosome, and then to carry out continuous transformation for inducing the modification of another target gene to the yeast strain.
    • 本发明涉及一种用于酵母转化的新型双组分表达载体,以及使用其的靶核苷酸序列的转化方法。 本发明的表达模块包括:表达模块,其包含多克隆位点(MCS),适当的启动子和终止子; 和救援模块。 本发明的载体能够稳定地表达酵母,特别是马克斯克鲁维酵母(Kluyveromyces marxianus)或酿酒酵母(Saccharomyces cerevisiae)中的靶基因,能够高效地将靶基因整合到染色体的特定位置。 使用本发明的载体的转化方法可以非常有用地应用于所需产物(生物质)的工业生产,因为可以制备靶基因稳定整合到染色体中的转基因酵母菌株,然后携带 用于诱导酵母菌株的另一靶基因的修饰的连续转化。
    • 8. 发明申请
    • SACCHAROMYCES-CEREVISIAE-HEFESTAMM MIT STABILER INTEGRATION UND EXPRESSION DER NUKLEINSÄURESEQUENZ FÜR EINEN HETEROLOGEN KALIUMIONEN-KANAL
    • 酿酒酵母酵母菌株稳定整合AND NUCLEIC序列的表达异源钾离子通道
    • WO2003031600A1
    • 2003-04-17
    • PCT/EP2001/011488
    • 2001-10-05
    • LICHTENBERG-FRATÉ, HellaLUDWIG, Jost
    • LICHTENBERG-FRATÉ, HellaLUDWIG, Jost
    • C12N1/19
    • C12R1/865C07K14/705C12N15/905G01N33/6872
    • Die Erfindung betrifft Saccharomyces-cerevisiae-Hefestämme mit stabiler Integration und Expression der Nukleinsäuresequenz für einen heterologen Kaliumionen-Kanal sowie das Verfahren zur Konstruktion dieser Hefestämme. Im Einzelnen betrifft die Erfindung einen Saccharomyces-cerevisiae-Hefestamm, in dem alle identifizierten Kaliumionentranslokations-systeme spezifisch deletiert sind; einen solchen Saccharomyces-cerevisiae-Hefestamm, in den darüber hinaus eine Nukleinsäuresequenz, die für einen heterologen Kaliumionen-Kanal, insbesondere für einen Säuger-Kaliumionen-Kanal wie den humanen erg-Kaliumionen-Kanal (HERG) kodiert, stabil in das Hefegenom integriert ist und exprimiert wird; ein Verfahren zur stabilen Integration heterologer Kaliumionen-Kanalproteine in einen Saccharomyces-cerevisiae-Hefestamm sowie ein Verfahren zur Detektion spezifischer Modulatoren von Säuger-Kaliumionen-Kanälen unter Verwendung der genannten Hefestämme.
    • 本发明涉及一种具有稳定整合和核酸序列的表达异源钾离子通道,以及用于此酵母菌株施工方法酿酒酵母酵母菌株。 更具体地说,本发明涉及一种酿酒酵母酵母菌株,其中所有系统被识别Kaliumionentranslokations具体删除; 这样的酿酒酵母酵母菌株,其进一步包括用于哺乳动物钾离子通道作为人ERG钾离子通道(HERG)编码异源钾离子通道的核酸序列,特别是稳定地整合到在酵母基因组 并表达; 用于异源钾离子通道蛋白的在酿酒酵母酵母菌株中稳定整合和用于检测使用所述酵母菌株哺乳动物钾离子通道的特异性调节剂的方法的方法。
    • 9. 发明申请
    • CELLS ENGINEERED TO CONTAIN GENES OF INTEREST WITHOUT DRUG RESISTANCE GENE MARKERS AND MATERIALS AND METHODS THEREFOR
    • 细胞工程含有不具有耐药基因标记的物质基因及其材料及方法
    • WO02029009A1
    • 2002-04-11
    • PCT/US2001/030912
    • 2001-10-03
    • C12N15/09C12N1/19C12N15/81C12N15/90C12P7/18C12P9/00C12R1/85C12N1/20C12N1/14C12N1/16C12N1/18C12N15/00C12N15/63C12N15/70
    • C12P9/00C12N15/81C12N15/905
    • Methods and materials are provided for stably introducing any gene into a specific locus in the genome of a microorganism such as yeast without the addition of any drug resistance genes. Specifically provided herein are new genetically engineered inositol-overproducing Saccharomyces cerevisiae strains obtained by using a novel set of yeast integration plasmids that allow the safe, stable, and controlled introduction of homologous as well as heterologous genes into the host genome. In particular, specific loci of the S. cervisiae yeast genome can be targeted with single or multiple copies of a specific gene that is desired to be expressed or a given set of specific genes that the host can use without the addition of any drug resistance genes. The principles of this new methodology can also be used for the construction of other recombinant yeast and bacterial strains as well as higher eukaryotic cells.
    • 提供方法和材料用于将任何基因稳定地引入微生物如酵母的基因组中的特定基因座,而不添加任何耐药性基因。 本文具体提供的是新的遗传工程肌醇过量产生的酿酒酵母菌株,其通过使用新的酵母整合质粒组来获得,所述质粒允许将同源和异源基因安全,稳定和控制地引入宿主基因组。 特别地,S.Carvisiae酵母基因组的特定位点可以靶向希望表达的特定基因的单拷贝或多个拷贝,或宿主可以使用而不添加任何药物抗性基因的给定的一组特定基因 。 这种新方法的原理也可用于构建其他重组酵母和细菌菌株以及高等真核细胞。
    • 10. 发明申请
    • METHODS FOR PRODUCING A POLYPEPTIDE USING A CRIPPLED TRANSLATIONAL INITIATOR SEQUENCE
    • 使用切片翻译序列产生多肽的方法
    • WO0151646A3
    • 2002-02-14
    • PCT/US0101102
    • 2001-01-12
    • NOVOZYMES BIOTECH INC
    • YAVER DEBBIE SBELLINI DANIEL A
    • C12N15/80C12N15/90C12N15/67C12N1/15C12N15/65
    • C12N15/905C12N15/80
    • The present invention relates to methods for producing a polypeptide, comprising: (a) cultivating a fungal host cell in a medium conducive for the production of the polypeptide; and (b) isolating the polypeptide from the cultivation medium; wherein the fungal host cell comprises a first nucleic acid sequence encoding the polypeptide in tandem with a second nucleic acid sequence comprising a crippled translational initiator sequence operably linked to a gene encoding a selectable marker, wherein the copy number of the first nucleic acid sequence has been increased by culturing the cell under conditions that select for multiple copies of the selectable marker. The present invention also relates to such fungal host cells and methods for obtaining such fungal host cells. The present invention further relates to nucleic acid constructs and vectors comprising a crippled translational initiator sequence operably linked to a gene encoding a selectable marker.
    • 本发明涉及产生多肽的方法,包括:(a)在有助于产生多肽的培养基中培养真菌宿主细胞; 和(b)从培养基中分离多肽; 其中所述真菌宿主细胞包含编码所述多肽的第一核酸序列与第二核酸序列串联,所述第二核酸序列包含可操作地连接到编码可选择标记的基因的残基翻译起始序列,其中所述第一核酸序列的拷贝数已被 通过在选择可选择标记的多个拷贝的条件下培养细胞而增加。 本发明还涉及这种真菌宿主细胞和获得这种真菌宿主细胞的方法。 本发明还涉及核酸构建体和载体,其包含与编码可选择标记的基因可操作地连接的残基翻译起始序列。