WO2020000461A1 - CRISPR/Cas9 靶向敲除人 NARC1 基因的方法及其特异性 gRNA - Google Patents

CRISPR/Cas9 靶向敲除人 NARC1 基因的方法及其特异性 gRNA Download PDF

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WO2020000461A1
WO2020000461A1 PCT/CN2018/093871 CN2018093871W WO2020000461A1 WO 2020000461 A1 WO2020000461 A1 WO 2020000461A1 CN 2018093871 W CN2018093871 W CN 2018093871W WO 2020000461 A1 WO2020000461 A1 WO 2020000461A1
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narc1
human
gene
grna
crispr
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毛吉炎
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深圳市博奥康生物科技有限公司
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    • 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

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  • the invention belongs to the field of molecular biology, and particularly relates to a CRISPR / Cas9 targeted knockout method of human NARC1 gene and a specific gRNA thereof.
  • LDL-C Low-density lipoprotein cholesterol
  • LDLR plasma low-density lipoprotein cholesterol receptor
  • Human NARC1 protein can directly increase the degradation of LDLR in the nucleus or lysosome and reduce the amount of LDLR, thereby increasing the amount of LDL-C circulation.
  • the NARC1 gene belongs to the preprotein converting enzyme (PC) family, which can directly increase the degradation of LDLR in the nucleus or lysosome and reduce the amount of LDLR, thereby increasing the amount of LDL-C circulation; NARC1 can also reduce the amount of LDLR entering the cytosol.
  • PC preprotein converting enzyme
  • indirectly increasing the plasma concentration of LDL-C can be used as a therapeutic target for high cholesterol-related cardiovascular and cerebrovascular diseases, and a large amount of research is needed to achieve clinical transformation.
  • the lack of a means to target the NARC1 gene expression in the prior art has caused a certain obstacle to the progress of related research.
  • the object of the present invention is to provide a method for targeting knockout of human NARC1 gene and its specific gRNA through design, construction and detection.
  • the present invention uses the CRISPR / Cas9 system principle and gRNA design principles as the basis, and uses software to design gRNA to target the human NARC1 gene.
  • the forward and reverse complementary oligonucleotide strands corresponding to the gRNA were synthesized, double stranded after annealing, and then connected to the px459 vector to construct a recombinant plasmid.
  • the recombinant plasmid was transfected into HPA-s cells for target site knockout verification.
  • the target sequence of the gRNA in the human NARC1 gene conforms to the 5'-N (20) -NGG-3 'or 5'-CCN-N (20) -3' sequence arrangement rule;
  • the gRNA is unique in the target sequence of the human NARC1 gene.
  • a method for CRISPR / Cas9 targeted knockout of human NARC1 gene includes the following steps:
  • the px459 vector was digested with BbsI and ligated with the double-stranded gRNA oligonucleotide chain.
  • the ligation product was transformed into E. coli Stbl3 competent cells, and positive clones were selected on ampicillin-resistant plates, followed by sequencing and identification after expansion. ;
  • Lipofectamine 2000 transfection reagent was used to transfect plasmid px459-gRNA into HPA-s cells to achieve targeted knockout of human NARC1 gene. Then the genomic DNA of the cells was extracted for PCR amplification, and the PCR products were digested with T7 endonuclease I, and the results were confirmed by electrophoresis.
  • the gRNA provided by the present invention can achieve specific knockout of the human NARC1 gene, and is of great significance for studying the clinical treatment of diseases related to the human NARC1 gene.
  • FIG. 1 is a graph of T7 endonuclease I test results of cells in an experimental group and a control group.
  • the sequence of human NARC1 gene was found in GenBank, and gRNA was designed using online software. Add a G to the 5 'end of the forward oligonucleotide strand of the gRNA, and a C to the 3' end of the reverse oligonucleotide strand accordingly. At the same time, CACC was added to the 5 ′ end of the forward oligonucleotide strand of each pair of complementary sequences, and AAAC was added to the 5 ′ end of the reverse oligonucleotide strand. The ends formed after annealing were digested with the px459 vector and digested with Bbs I. The sticky ends formed later are complementary.
  • the oligonucleotide chain was diluted to a final concentration of 100 ⁇ mol / L, and an annealing reaction was performed.
  • the reaction system is as follows: two complementary Oligo DNAs 0.5 ⁇ l each, 2 ⁇ l Annealing Buffer (10 ⁇ ), 17 ⁇ l ddH2O. After centrifuging the above system for an instant, place it in a boiling water bath and incubate for 5 min, then take it out and slowly cool it to room temperature for 1-2 hours.
  • the px459 vector was digested with Bbs I enzyme (Fermentas), the target fragment was purified and recovered, and ligated with the gRNA double-stranded DNA to construct a recombinant plasmid px459-gRNA.
  • the ligation reaction system is as follows: 2 ⁇ l gRNA double-stranded DNA, 2 ⁇ l px459 digested fragments, 1 ⁇ l T4 DNA ligase buffer, 1 ⁇ l T4 DNA ligase (NEB), 4 ⁇ l ddH2O. After centrifuging the above system transiently, it was incubated in a 16 ° C water bath overnight. The ligation product was transformed into E.
  • Embodiment 4 T7 Endonuclease I Examine transfection results
  • the transfected HPA-s cells (experimental group) and normal HPA-s cells (control group) were expanded and their genomic DNAs were extracted and amplified by high-fidelity PCR.
  • the PCR product was recovered by electrophoresis, and the product was digested with T7 endonuclease I.
  • the reaction system was: 1 ⁇ l 10 ⁇ T7E1 Buffer, 1 ⁇ l T7 endonuclease I, 1 ⁇ g of the recovered PCR product, made up to 10 with water. ⁇ l.
  • the reaction conditions were: 37 ° C water bath for 1 h. After the digestion, 1% agarose gel electrophoresis was performed, and the results are shown in FIG. 1. It can be seen that the PCR product of the control group was still only one band after digestion, while the experimental group showed multiple bands, indicating that the NARC1 gene in HPA-s cells was successfully edited.
  • the gRNA provided by the present invention can achieve specific knockout of the human NARC1 gene, and is of great significance for studying the clinical treatment of diseases related to the human NARC1 gene.

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Abstract

提供了一种CRISPR/Cas9靶向敲除人NARC1基因的方法及其特异性gRNA。所述方法根据CRISPR/Cas9的设计原则，在人NARC1基因的上下游设计两个靶位点，合成相应的寡核苷酸序列，并连接至px459载体上构建重组质粒，将其转染人食管癌细胞株，能够特异性敲除人NARC1基因，可应用于人NARC1基因相关疾病的临床治疗。

Description

CRISPR/Cas9靶向敲除人NARC1基因的方法及其特异性gRNA 技术领域
本发明属于分子生物学领域，具体涉及一种CRISPR/Cas9靶向敲除人NARC1基因的方法及其特异性gRNA。
 背景技术
人体内低密度脂蛋白胆固醇（LDL-C）主要是在细胞溶质中，通过与LDLR的结合来完成代谢。LDL-C的多少则主要取决于血浆低密度脂蛋白胆固醇受体（LDLR）的循环。人NARC1蛋白可以直接增加细胞核或者溶酶体内LDLR的降解，减少LDLR的量，从而增加LDL-C的循环量。
 技术问题
NARC1基因属于前蛋白转化酶(PC)家族，可以直接增加细胞核或者溶酶体内 LDLR 的降解，减少 LDLR 的量，从而增加 LDL-C的循环量；NARC1亦可以减少进入细胞溶质内 LDLR 的量，从而间接增加 LDL-C 的血浆浓度，可作为高胆固醇相关的心脑血管疾病的治疗靶点，需做大量研究方可实现临床转化。但现有技术中缺乏靶向敲除NARC1基因表达的手段，对相关研究的进展造成了一定的阻碍。
 技术解决方案
本发明的目的在于通过设计、构建和检测，提供靶向敲除人NARC1基因的方法及其特异性gRNA。
为实现上述目的，本发明以CRISPR/Cas9系统原理和gRNA设计原则为基础，利用软件设计gRNA，靶向人NARC1基因。合成gRNA相对应的正向和反向互补寡核苷酸链，退火后形成双链，连接至px459载体构建重组质粒；将重组质粒转染HPA-s细胞中进行靶位点敲除验证。
本发明申请的技术方案如下：
1、靶向人NARC1基因的gRNA设计，gRNA对应的寡核苷酸链的合成，携带gRNA寡核苷酸链的CRISPR/Cas9重组质粒构建。
2、在HPA-s细胞模型中分析鉴定本发明gRNA指导的CRISPR/Cas9系统对于靶向敲除人NARC1基因的特异性。
一种CRISPR/Cas9靶向敲除人NARC1基因的方法及其特异性gRNA：
a. 所述gRNA在人NARC1基因的靶序列符合5′-N(20)-NGG-3′或者5′-CCN-N(20)-3′序列的排列规则；
b. 所述gRNA在人NARC1基因的靶序列是唯一的。
上述一种CRISPR/Cas9靶向敲除人NARC1基因的方法及其特异性gRNA，其对应的DNA序列如序列表SEQ ID NO. 1所示。
一种CRISPR/Cas9靶向敲除人NARC1基因的方法，包含如下步骤：
a. 如权利要求1-2任意一项所述的gRNA，在gRNA序列SEQ ID NO. 1的互补链的5′端加上CACCG，合成得到正向寡核苷酸，在所述的SEQ ID NO. 1序列的5′端加上AAAC，3′端加上C，合成得到反向寡核苷酸链。将合成的一对互补正、反寡核苷酸链退火，形成双链gRNA寡核苷酸链；
b. 将px459载体经BbsⅠ酶切后，与上述双链gRNA寡核苷酸链连接，连接产物转化大肠杆菌Stbl3感受态细胞，在氨苄青霉素抗性平板上筛选阳性克隆，扩大培养后进行测序鉴定；
c. 采用Lipofectamine 2000转染试剂将质粒px459-gRNA转染至HPA-s细胞，实现人NARC1基因的靶向敲除。然后提取细胞基因组DNA进行PCR扩增，并用T7核酸内切酶I对PCR产物进行酶切，电泳确认酶切结果。
 有益效果
本发明提供的gRNA能够实现人NARC1基因的特异性敲除，对于研究与人NARC1基因相关疾病的临床治疗具有重要意义。
 附图说明
图1为实验组和对照组细胞的T7核酸内切酶I试验结果图。
 本发明的实施方式
下面结合具体实施例和附图进一步阐述本发明。
实施例一：靶向人 NARC1 基因的 gRNA 设计及寡核苷酸链的合成
在GenBank中找到人NARC1基因的序列，利用在线软件设计gRNA。在gRNA正向寡核苷酸链的5′端添加一个G，相应地在反向寡核苷酸链的3′端添加一个C。同时在每对互补序列的正向寡核苷酸链的5′端添加CACC，反向寡核苷酸链的5′端添加AAAC，使其退火后形成的末端与px459载体经Bbs Ⅰ酶切后形成的粘性末端互补。
实施例二：靶向人 NARC1 基因增的 CRISPR/Cas9 重组质粒构建
将上述寡核苷酸链稀释至终浓度为100μmol/L，进行退火反应。反应体系如下：两条互补Oligo  DNA各0.5μl，2μl  Annealing  Buffer(10×)，17 μl  ddH2O。将以上体系瞬时离心后，置于沸水浴中孵育5 min，随后取出，缓慢冷却1～2h至室温。
用Bbs I酶（Fermentas）对px459载体进行酶切，纯化回收目的片段，与上述gRNA双链DNA连接，构建重组质粒px459-gRNA。连接反应体系如下：2 μl gRNA双链DNA，2 μl px459酶切片段，1 μl T4 DNA连接酶buffer，1μl T4 DNA 连接酶（NEB），4μl ddH2O。将以上体系瞬时离心后，置于16℃水浴中孵育过夜。连接产物转化大肠杆菌Stbl3感受态细胞，在氨苄青霉素抗性平板上筛选阳性克隆，扩大培养后进行测序鉴定。对于测序结果正确的阳性克隆，无内毒素提取其中的重组质粒px459-gRNA。
实施例三：重组质粒 px459-gRNA 的转染
将HPA-s西部接种于六孔板，每孔加入含10% FBS的DMEM培养基培养2 ml进行培养，18-24 h后细胞汇合率达50％～60％时即可用于转染。参照Lipofectamine 2000转染试剂说明采用脂质体法转染重组质粒px459-gRNA。
实施例四： T7 核酸内切酶 I 检验转染结果
扩大培养经转染的HPA-s细胞（实验组）和正常的HPA-s细胞（对照组），分别提取其基因组DNA后，高保真PCR扩增。电泳回收PCR产物，然后用T7核酸内切酶I对产物进行酶切，反应体系为：1 μl 10 × T7E1 Buffer，1 μl T7核酸内切酶I，1 μg 回收的PCR产物，用水补足至10 μl。反应条件为：37℃水浴1 h。酶切结束后进行1%琼脂糖凝胶电泳，结果如图1所示。可以看到，对照组细胞的PCR产物经酶切后仍然只有1条带，而实验组则出现了多条带，说明HPA-s细胞中的NARC1基因被成功编辑。
 工业实用性
本发明提供的gRNA能够实现人NARC1基因的特异性敲除，对于研究与人NARC1基因相关疾病的临床治疗具有重要意义。

Claims (3)

  1. 一种CRISPR/Cas9靶向敲除人NARC1基因的方法及其特异性gRNA，其特征为：
    a. 所述gRNA在人NARC1基因的靶序列符合5′-N(20)-NGG-3′或者5′-CCN-N(20)-3′序列的排列规则；
    b. 所述gRNA在人NARC1基因的靶序列是唯一的。
  2. 根据权利要求1所述的一种CRISPR/Cas9靶向敲除人NARC1基因的方法及其特异性gRNA，其特征在于：其对应的DNA序列如序列表SEQ ID NO. 1所示。
  3. 一种CRISPR/Cas9靶向敲除人NARC1基因的方法，其特征在于，包含如下步骤：
    a. 如权利要求1-2任意一项所述的gRNA，在gRNA序列SEQ ID NO. 1的互补链的5′端加上CACCG，合成得到正向寡核苷酸，在所述的SEQ ID NO. 1序列的5′端加上AAAC，3′端加上C，合成得到反向寡核苷酸链。将合成的一对互补正、反寡核苷酸链退火，形成双链gRNA寡核苷酸链；
    b. 将px459载体经BbsⅠ酶切后，与上述双链gRNA寡核苷酸链连接，连接产物转化大肠杆菌Stbl3感受态细胞，在氨苄青霉素抗性平板上筛选阳性克隆，扩大培养后进行测序鉴定；
    c. 采用Lipofectamine 2000转染试剂将质粒px459-gRNA转染至HPA-s细胞，实现人NARC1基因的靶向敲除。然后提取细胞基因组DNA进行PCR扩增，并用T7核酸内切酶I对PCR产物进行酶切，电泳确认酶切结果。
PCT/CN2018/093871 2018-06-29 2018-06-29 CRISPR/Cas9 靶向敲除人 NARC1 基因的方法及其特异性 gRNA WO2020000461A1 (zh)

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Citations (2)

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CN103820441A (zh) * 2014-03-04 2014-05-28 黄行许 CRISPR-Cas9特异性敲除人CTLA4基因的方法以及用于特异性靶向CTLA4基因的sgRNA
CN105886498A (zh) * 2015-05-13 2016-08-24 沈志荣 CRISPR-Cas9特异性敲除人PCSK9基因的方法以及用于特异性靶向PCSK9基因的sgRNA

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CN103820441A (zh) * 2014-03-04 2014-05-28 黄行许 CRISPR-Cas9特异性敲除人CTLA4基因的方法以及用于特异性靶向CTLA4基因的sgRNA
CN105886498A (zh) * 2015-05-13 2016-08-24 沈志荣 CRISPR-Cas9特异性敲除人PCSK9基因的方法以及用于特异性靶向PCSK9基因的sgRNA

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