WO2021169457A1 - Arnsg de gène de résistance à la tétracycline de ciblage teta, son vecteur d'inactivation, procédé d'établissement de vecteur et application associée - Google Patents

Arnsg de gène de résistance à la tétracycline de ciblage teta, son vecteur d'inactivation, procédé d'établissement de vecteur et application associée Download PDF

Info

Publication number
WO2021169457A1
WO2021169457A1 PCT/CN2020/132997 CN2020132997W WO2021169457A1 WO 2021169457 A1 WO2021169457 A1 WO 2021169457A1 CN 2020132997 W CN2020132997 W CN 2020132997W WO 2021169457 A1 WO2021169457 A1 WO 2021169457A1
Authority
WO
WIPO (PCT)
Prior art keywords
sgrna
teta
gene
vector
cas9
Prior art date
Application number
PCT/CN2020/132997
Other languages
English (en)
Chinese (zh)
Inventor
陈红
林泽俊
朱琳
周振超
Original Assignee
浙江大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 浙江大学 filed Critical 浙江大学
Publication of WO2021169457A1 publication Critical patent/WO2021169457A1/fr

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]

Definitions

  • primer pair 1 PCR was used to amplify the RP4-oriT sequence, where the RP4-oriT sequence contained the conjugative transfer site, and the amplified product was gel purified.
  • the oriV-oriT-sgRNA (tetA) sequence is amplified by primer pair 4 PCR, and the amplified product is gel purified.
  • Figure 1 is a map of puc57-sgRNA (tetA) plasmid in Example 2 of the present invention. The figure shows the replicon (ori), the ampicillin resistance gene (Amp), and the expression element sgRNA (tetA) of sgRNA-1.
  • Example 2 The transformation steps involved in Example 2 are as follows: take 100ul of competent cells to thaw on ice, add the seamless clone ligation product, and place on ice for 30 minutes; put the competent cells in a 42°C water bath for 60 seconds and quickly transfer to an ice bath Cool for 3 minutes; add 500ul LB liquid medium to the competent cells, mix well and place in a 200rpm shaker at 37°C to recover for 60 minutes; draw 100ul of the recovered competent cells and spread on the corresponding resistant medium , Placed in a constant temperature incubator for 16 hours.
  • Control group Add 100ng puc57-oriT-Cas9- ⁇ sgRNA control vector to 50ul competent cells and place on ice for 30 minutes.

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Mycology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un ARNsg d'un gène de résistance aux antibiotiques de ciblage tetA spécifique, un vecteur d'invalidation génique CRISPR/Cas9 contenant l'ARNsg, un procédé d'établissement du vecteur, et une application associée, c'est-à-dire un ARNsg d'un gène de résistance à la tétracycline de ciblage tetA, un vecteur d'inactivation associé, un procédé d'établissement du vecteur, et une application associée. La séquence de l'ARNsg qui effectue une coupe ciblée sur le gène tetA est criblée au moyen de l'analyse de séquence sur le gène de résistance à la tétracycline tetA. Des expériences vérifient que le vecteur d'inactivation CRISPR/Cas9 selon l'invention peut effectuer efficacement une coupe ciblée sur les séquences 251 à 270 du gène tetA.
PCT/CN2020/132997 2020-02-29 2020-11-30 Arnsg de gène de résistance à la tétracycline de ciblage teta, son vecteur d'inactivation, procédé d'établissement de vecteur et application associée WO2021169457A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010132908.3 2020-02-29
CN202010132908.3A CN111378660B (zh) 2020-02-29 2020-02-29 一种靶向四环素抗性基因tetA的sgRNA及其敲除载体、载体构建方法和应用

Publications (1)

Publication Number Publication Date
WO2021169457A1 true WO2021169457A1 (fr) 2021-09-02

Family

ID=71217086

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/132997 WO2021169457A1 (fr) 2020-02-29 2020-11-30 Arnsg de gène de résistance à la tétracycline de ciblage teta, son vecteur d'inactivation, procédé d'établissement de vecteur et application associée

Country Status (2)

Country Link
CN (1) CN111378660B (fr)
WO (1) WO2021169457A1 (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111378660B (zh) * 2020-02-29 2021-08-06 浙江大学 一种靶向四环素抗性基因tetA的sgRNA及其敲除载体、载体构建方法和应用
US20240016855A1 (en) * 2020-10-13 2024-01-18 Centre National De La Recherche Scientifique (Cnrs) Targeted-antibacterial-plasmids combining conjugation and crispr/cas systems and uses thereof
CN113817731A (zh) * 2021-07-31 2021-12-21 浙江大学 一种靶向削减耐药基因blaTEM及其耐药质粒的gRNA、可转移型敲除载体及其应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150064138A1 (en) * 2013-09-05 2015-03-05 Massachusetts Institute Of Technology Tuning microbial populations with programmable nucleases
KR101584933B1 (ko) * 2015-02-10 2016-01-13 성균관대학교산학협력단 항생제 내성 억제용 재조합 벡터 및 이의 용도
CN106536739A (zh) * 2014-04-14 2017-03-22 内梅西斯生物有限公司 治疗剂
CN106636355A (zh) * 2016-11-17 2017-05-10 重庆高圣生物医药有限责任公司 四环素耐药基因检测并联探针、基因芯片、试剂盒与检测法
CN107384926A (zh) * 2017-08-13 2017-11-24 中国人民解放军疾病预防控制所 一种靶向清除细菌耐药性质粒的CRISPR‑Cas9系统及应用
CN111378660A (zh) * 2020-02-29 2020-07-07 浙江大学 一种靶向四环素抗性基因tetA的sgRNA及其敲除载体、载体构建方法和应用

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102719431B (zh) * 2012-06-20 2013-06-19 浙江大学 一种检测污泥中四环素类抗性基因tetB的引物序列及方法
US9988637B2 (en) * 2015-10-26 2018-06-05 National Tsing Hua Univeristy Cas9 plasmid, genome editing system and method of Escherichia coli
CN106191116B (zh) * 2016-08-22 2019-10-08 西北农林科技大学 基于CRISPR/Cas9的外源基因敲入整合系统及其建立方法和应用
CN108220329A (zh) * 2017-12-29 2018-06-29 四川省农业科学院生物技术核技术研究所 一种敲除转基因植物中潮霉素抗性基因的方法
CN109486844B (zh) * 2018-10-12 2020-08-25 中南民族大学 一种肠毒素性大肠杆菌的特异性标记方法
CN110066829B (zh) * 2019-04-30 2023-04-28 江南大学 一种CRISPR/Cas9基因编辑系统及其应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150064138A1 (en) * 2013-09-05 2015-03-05 Massachusetts Institute Of Technology Tuning microbial populations with programmable nucleases
CN106536739A (zh) * 2014-04-14 2017-03-22 内梅西斯生物有限公司 治疗剂
KR101584933B1 (ko) * 2015-02-10 2016-01-13 성균관대학교산학협력단 항생제 내성 억제용 재조합 벡터 및 이의 용도
CN106636355A (zh) * 2016-11-17 2017-05-10 重庆高圣生物医药有限责任公司 四环素耐药基因检测并联探针、基因芯片、试剂盒与检测法
CN107384926A (zh) * 2017-08-13 2017-11-24 中国人民解放军疾病预防控制所 一种靶向清除细菌耐药性质粒的CRISPR‑Cas9系统及应用
CN111378660A (zh) * 2020-02-29 2020-07-07 浙江大学 一种靶向四环素抗性基因tetA的sgRNA及其敲除载体、载体构建方法和应用

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DAMIEN JACOT, DOMINIQUE SOLDATI: "CRISPR/Cas9-Mediated Generation of Tetracycline Repressor-Based Inducible Knockdown in Toxoplasma gondii.", METHODS IN MOLECULAR BIOLOGY (TOXOPLASMA GONDII : METHODS AND PROTOCOLS), 23 November 2019 (2019-11-23), US, pages 125 - 141, XP009530036, ISSN: 1064-3745, ISBN: 978-1-60761-961-1, DOI: 10.1007/978-1-4939-9857-9_7 *
QIU HAIXIANG, GONG JIANSEN, BUTAYE PATRICK, LU GUANGWU, HUANG KE, ZHU GUOQIANG, ZHANG JILEI, HATHCOCK TERRI, CHENG DARONG, WANG CH: "CRISPR/Cas9/sgRNA-mediated targeted gene modification confirms the cause-effect relationship between gyrA mutation and quinolone resistance in Escherichia coli", FEMS MICROBIOLOGY LETTERS, vol. 365, no. 13, 1 July 2018 (2018-07-01), pages 127, XP055841701, DOI: 10.1093/femsle/fny127 *
TAO WANBING: "Toxicity Evaluation of Sulfonamides to Gene Knock-Down Escherichia coli Based on CRISPR-dCas9 System", CHINESE MASTER’S THESES FULL-TEXT DATABASE, MEDICINE & HEALTH SCIENCES, 15 September 2019 (2019-09-15), XP055841703 *
YOO KYUNG KANG, KYU KWON, JEA SUNG RYU, HA NEUL LEE, CHANKYU PARK, HYUN JUNG CHUNG: "Nonviral Genome Editing Based on a Polymer-Derivatized CRISPR Nanocomplex for Targeting Bacterial Pathogens and Antibiotic Resistance", BIOCONJUGATE CHEMISTRY, AMERICAN CHEMICAL SOCIETY, US, vol. 28, no. 4, 19 April 2017 (2017-04-19), US, pages 957 - 967, XP055647110, ISSN: 1043-1802, DOI: 10.1021/acs.bioconjchem.6b00676 *

Also Published As

Publication number Publication date
CN111378660B (zh) 2021-08-06
CN111378660A (zh) 2020-07-07

Similar Documents

Publication Publication Date Title
WO2021169457A1 (fr) Arnsg de gène de résistance à la tétracycline de ciblage teta, son vecteur d'inactivation, procédé d'établissement de vecteur et application associée
Tischer et al. En passant mutagenesis: a two step markerless red recombination system
CN107142272A (zh) 一种控制大肠杆菌中质粒复制的方法
CN107893080A (zh) 一种靶向大鼠Inhba基因的sgRNA及其应用
CN104673816A (zh) 一种pCr-NHEJ载体及其构建方法及其用于细菌基因定点敲除的应用
CN107904228B (zh) 一种基于热击的哈维弧菌同源重组基因敲除的方法
CN111607594A (zh) 一种基于CRISPR-Cas9编辑技术的敲除猪IRF8基因的细胞系及其构建方法
CN109825464B (zh) 敲除t6ss-1基因簇的杀香鱼假单胞菌鱼用减毒疫苗
CN110129228B (zh) 诺卡氏菌感受态细胞的制备方法与诺卡氏菌基因编辑方法
CN110591994B (zh) 一种基于氢氧化钠刺激的哈维弧菌同源重组基因敲除的方法
CN103966249B (zh) 一种用于构建无筛选标签蓝细菌的载体及其应用
JP6858191B2 (ja) 酵母細胞の相同的組み換えノックアウトのための遺伝子カセット
CN115976103B (zh) 一种双壳贝类生长调控基因的功能验证方法
CN111154801B (zh) 一种提高基因编辑效率的方法
CN116478260A (zh) 一种小麦糖转运蛋白、基因及其应用
CN115851798A (zh) pEcCas9-2.0质粒及其构建方法和应用
CN106701607B (zh) 在酵母菌中实现高准确率定点基因敲除的方法
CN109628361B (zh) 整合双拷贝功能性f4菌毛操纵子基因猪源益生菌ep1克隆株、构建方法及应用
CN108203697B (zh) 一种鱼类天然杀伤细胞增强因子的酵母工程菌及其应用
CN114540351B (zh) 靶向肺炎克雷伯菌MdtABC外排泵的sRNA及在制备四环素类抗生素耐药株中的应用
CN115976086B (zh) 一种细菌CRISPR-Cas9基因编辑的方法和应用
CN116218894A (zh) 一种少动鞘氨醇单胞菌CRISPRi系统、其构建方法及应用
CN107630010A (zh) 具有抑制细菌中质粒介导的喹诺酮类耐药基因qnrD的脱氧核酶及其应用
CN116121288B (zh) 一组克隆恶臭假单胞菌大片段dna的载体及其应用
CN115960733B (zh) 一种用于大片段dna组装的基因工程酵母菌、构建方法及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20921267

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20921267

Country of ref document: EP

Kind code of ref document: A1