CN107828786A - 靶向敲除PIK3CA基因的sgRNA及应用 - Google Patents
靶向敲除PIK3CA基因的sgRNA及应用 Download PDFInfo
- Publication number
- CN107828786A CN107828786A CN201710997196.XA CN201710997196A CN107828786A CN 107828786 A CN107828786 A CN 107828786A CN 201710997196 A CN201710997196 A CN 201710997196A CN 107828786 A CN107828786 A CN 107828786A
- Authority
- CN
- China
- Prior art keywords
- crispr
- pik3ca
- cas9
- sgrna
- knock out
- Prior art date
- Legal status (The legal status 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 status listed.)
- Pending
Links
- 101000605639 Homo sapiens Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform Proteins 0.000 title claims abstract description 30
- 108091027544 Subgenomic mRNA Proteins 0.000 title claims abstract description 26
- 230000008685 targeting Effects 0.000 title claims abstract description 24
- 210000004027 cell Anatomy 0.000 claims abstract description 23
- 206010006187 Breast cancer Diseases 0.000 claims abstract description 17
- 208000026310 Breast neoplasm Diseases 0.000 claims abstract description 17
- 230000014509 gene expression Effects 0.000 claims abstract description 17
- 108091028043 Nucleic acid sequence Proteins 0.000 claims abstract description 8
- 210000004881 tumor cell Anatomy 0.000 claims abstract description 5
- 238000010356 CRISPR-Cas9 genome editing Methods 0.000 claims description 26
- 230000029087 digestion Effects 0.000 claims description 11
- 102100038332 Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform Human genes 0.000 claims description 9
- 101150063858 Pik3ca gene Proteins 0.000 claims description 9
- 108090000623 proteins and genes Proteins 0.000 claims description 7
- 210000002919 epithelial cell Anatomy 0.000 claims description 6
- 239000000969 carrier Substances 0.000 claims description 5
- 239000003292 glue Substances 0.000 claims description 5
- 201000008275 breast carcinoma Diseases 0.000 claims description 4
- 238000010276 construction Methods 0.000 claims description 4
- 238000001890 transfection Methods 0.000 claims description 4
- 102000004190 Enzymes Human genes 0.000 claims description 3
- 108090000790 Enzymes Proteins 0.000 claims description 3
- 101001091385 Homo sapiens Kallikrein-6 Proteins 0.000 claims description 3
- 102100034866 Kallikrein-6 Human genes 0.000 claims description 3
- 108091007960 PI3Ks Proteins 0.000 abstract description 8
- 102000010400 1-phosphatidylinositol-3-kinase activity proteins Human genes 0.000 abstract description 7
- 239000013612 plasmid Substances 0.000 abstract description 7
- 238000011160 research Methods 0.000 abstract description 6
- 108091033409 CRISPR Proteins 0.000 abstract description 4
- 230000010534 mechanism of action Effects 0.000 abstract 1
- 239000012634 fragment Substances 0.000 description 8
- 108020004414 DNA Proteins 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 7
- 238000010362 genome editing Methods 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 5
- 206010028980 Neoplasm Diseases 0.000 description 5
- 101150038500 cas9 gene Proteins 0.000 description 5
- 230000008901 benefit Effects 0.000 description 4
- 229920000936 Agarose Polymers 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 230000009456 molecular mechanism Effects 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 108020005004 Guide RNA Proteins 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- 229920000297 Rayon Polymers 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 230000001629 suppression Effects 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 208000003174 Brain Neoplasms Diseases 0.000 description 1
- 238000010354 CRISPR gene editing Methods 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 239000012097 Lipofectamine 2000 Substances 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 108090000430 Phosphatidylinositol 3-kinases Proteins 0.000 description 1
- 102000003993 Phosphatidylinositol 3-kinases Human genes 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 239000000686 essence Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
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
- C07K14/82—Translation products from oncogenes
-
- 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
- C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
-
- 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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Oncology (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Mycology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
本发明公开了一种靶向敲除PIK3CA基因的sgRNA及应用,其具有下列核苷酸序列之一:a、sgRNA1:TCCGCGGCTCTAACCGCATCGGG;b、sgRNA2:ACCCGATGCGGTTAGAGCCGCGG;该sgRNA对PIK3CA基因的切割效率高;将含有该sgRNA的CRISPR‑Cas9系统质粒转染到乳腺癌SK‑BR‑3细胞株中,得到的细胞株PI3K蛋白表达水平显著降低。因此,本发明提供的sgRNA能有效靶向敲除PIK3CA基因,从而有利于研究细胞株中PI3K低表达后的作用机制,为以PIK3CA基因为靶点的肿瘤细胞研究做出重要贡献。
Description
技术领域
本发明涉及生物技术领域,特别涉及一种靶向敲除PIK3CA基因的sgRNA及应用。
背景技术
PIK3CA基因定位于3q26.3,长34kb,包含20个外显子。目前普遍认为PIK3CA是一种癌基因,其突变在结肠癌、脑癌、乳腺癌、肺癌等多种实体肿瘤中均有报道。PIK3CA编码Ⅰ类磷脂酰肌醇-3-激酶的p110催化亚单位,即PI3Kp110α。现主流学术认为PIK3CA突变主要通过PI3K/AKT通路影响肿瘤的发生发展,而由于肿瘤的异质性,发现PIK3CA突变后影响下游的方式又不全都是通过这条通路,所以对其PIK3CA突变后下游通路的调控、激发的分子机制还需进一步的深入探索。
乳腺癌是恶性肿瘤之一,位居女性癌症发病之首,全球每年新发乳腺癌病例约167.1万,每年约52.2万人死于乳腺癌,严重危害了人类的生命健康。目前,乳腺癌的发生机制上已经做了大量研究,但发生与进展中涉及的分子机制仍有许多尚未明确。进一步明确乳腺癌发生发展的分子机制、寻找新的治疗靶点以及探索科学的综合治疗模式以提高早诊断早治疗,是乳腺癌研究的重要内容。
CRISPR-Cas9基因编辑技术是通过一段短的引导RNA ( guide RNA) 来识别特定的DNA 序列,后由其引导的Cas9蛋白定位到该特定的DNA序列上进行切割,从而起到基因编辑的作用。CRISPR-Cas9作为第三代基因编辑技术,其相对于前两代基因编辑技术具有系统构建简单、精准度高、成本低、能同时对多个位点进行定点编辑的各项优势,已成为目前研究的热点。为获得该技术基因编辑的优势作用,重点在于sgRNA(smallguide RNA)的设计。
目前,在研究乳腺癌的相关基因仍然均使用RNA干扰技术,其主要缺点是在RNA水平的干扰,效率低下,不适用于长期抑制研究。
发明内容
本发明目的在于提供一种靶向敲除PIK3CA基因的sgRNA,其具有下列核苷酸序列之一:
a、sgRNA:TCCGCGGCTCTAACCGCATCGGG(SEQ ID NO:1);
b、sgRNA2:ACCCGATGCGGTTAGAGCCGCGG(SEQ ID NO:2)。
该sgRNA可用于CRISPR-Cas9基因编辑系统中有效且高效敲低PIK3CA基因表达;该方法利用设计的特异sgRNA,精确指导cas9蛋白靶向切割PIK3CA基因。
本发明的另一个目的在于提供一种靶向敲除PIK3CA基因的CRISPR-Cas9系统,该系统含有上述靶向敲除PIK3CA基因的sgRNA的DNA序列。
上述靶向敲除PIK3CA基因的CRISPR-Cas9系统的构建方法包括如下步骤:
(1)用下面的引物扩增sgRNA1和sgRNA2序列:
CRISPR-F:5’-GTATTTCGATTTCTTGGCTTTATATATCT-3’ (SEQ ID NO:3);
CRISPR-R:5’-GTTGATAACGGACTAGCCTTATTTTAC-3’ (SEQ ID NO:4);
(2)使用XmaⅠ和PmeⅠ内切酶双酶切载体PENTY-U6-EF1a-Cas9,将双酶切产物通过跑1%的琼脂糖凝胶,切下含有目的条带胶块,使用QIAGEN的Gel Extraction kit进行胶回收得到酶切后的CRISPR-Cas9载体;
(3)使用Vazyme公司的clon Express Ⅱ one step cloning kit,将上述靶向敲除PIK3CA基因的sgRNA的PCR产物与酶切胶回收的CRISPR-Cas9载体连接,得到靶向敲除PIK3CA基因的CRISPR-Cas9系统。
本发明另一目的在于将靶向敲除PIK3CA基因表达的CRISPR-Cas9系统应用在制备敲除PIK3CA基因的细胞株中,即将靶向敲除PIK3CA基因的CRISPR-Cas9系统转染至目的细胞株中。
其中目的细胞株为肿瘤细胞株。
所述肿瘤细胞株为乳腺癌细胞株。
所述乳腺癌细胞株为人乳腺上皮细胞SK-BR-3。
本发明相对于现有技术具有如下的优点及效果:
(1)本发明提供靶向敲低PIK3CA基因表达的sgRNA能有效靶向PIK3CA基因,将其构建入CRISPR-Cas9系统中,转染入细胞可得到低表达的细胞株。其相对于前两代基因编辑技术此系统构建方法简单、成本低、可操作性强;
(2)目前,用于降低蛋白表达常使用RNA干扰技术,其主要缺点是在RNA水平的干扰效率低下,不适用于长期抑制研究;而使用CRISPR-Cas9系统可以构建出稳定低表达蛋白的细胞;在科研研究癌症发生与进展中所涉及的分子机制中有很大的优势。
附图说明
图1是用CRISPR-F、CRISPR-R引物分别扩增PIK3CAsgRNA1和PIK3CAsgRNA2序列后跑出的琼脂糖电泳图;
图2是双酶切后CRISPR-Cas9载体电泳图;
图3是PCR鉴定时,用鉴定引物P构建的CRISPR-Cas9载体系统的产物的琼脂糖电泳图;
图4是细胞株中PI3K蛋白的表达检测图;其中SK-BR-3-WT代表未转染CRISPR-Cas9载体系统的野生型乳腺上皮细胞株,SK-BR-3-sg1代表转染带有PIK3CAsgRNA1的CRISPR-Cas9载体系统的乳腺癌上皮细胞株,SK-BR-3-sg2代表转染带有PIK3CAsgRNA2的CRISPR-Cas9载体系统的乳腺癌上皮细胞株。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述。以下实施例仅用于说明本发明而不用于限制本发明的范围,该领域的技术人员可以根据上述本发明内容对发明做出一些非本质的改进和调整。文中未注明具体条件的实验方法,通常按照《分子克隆实验指南》中所述常规条件,或试剂制造厂商所建议的条件实施。除非另行定义,文中所使用的所以专业和科学用语与本领域熟练人员所熟悉的意义相同。
实验例1:使用CRISPR-Cas9技术构建敲低PIK3CA基因表达质粒
1、sgRNA寡核苷酸链合成
使用CRISPR在线设计工具(http://crispr.mit.edu/),输入PIK3CA的第1外显子序列,在给出的序列中,选择得分率高的sgRNA序列,后由生工公司合成。
表1 sgRNA寡核苷酸序列
;
2、用如下引物扩增sgRNA(sgRNA1或sgRNA2)片段
CRISPR-F:5’-GTATTTCGATTTCTTGGCTTTATATATCT-3’
CRISPR-R:5’-GTTGATAACGGACTAGCCTTATTTTAC-3’
引物稀释,将CRISPR-F、CRISPR-R引物用灭菌ddH2O稀释到终浓度为0.1μM,配置PCR反应体系为:
将上述试剂混匀后,上PCR仪,反应条件为:①94℃,5min预变性;②94℃,30s变性;③55℃,30s退火;④73℃延伸;循环②至④35次;⑤72℃,10mim;PCR产物跑琼脂糖凝胶验证片段大小,用D2000 marker标定,120bp处为扩增出的所需片段(图1)。
3、载体构建
(1)使用XmaⅠ和PmeⅠ两种内切酶双酶切PENTY-U6-EF1a-Cas9质粒,37℃水浴30min;
双酶切体系如下:
酶切产物跑1%的琼脂糖凝胶,用D2000 marker标定。双酶切后有23bp与8561bp两个片段,因23bp片段太短,无法在凝胶图上显示,我们需要的是8561bp的片段(图2);
(2)用刀片切下8561bp所在片段的胶块,使用QIAGEN胶回收试剂盒纯化酶切质粒产物,步骤按说明书进行操作;
(3)将步骤2 PCR扩增的120bp双链DNA产物和胶回收的酶切后CRISPR-Cas9质粒通过Vazyme公司的clon Express Ⅱ one step cloning kit进行重组连接,37℃,30min;
连接体系如下:
(4)将重组连接后的质粒转化至感受态细胞DH5α(TIANGEN)中,用250μl无抗性的液体培养基37℃摇床45min后,取50μl均匀涂至含有氨苄抗性的固体培养基平板上,置于37℃培养箱中培养12-16小时,菌落即可出现;
(5)挑取单个菌落扩大培养并质粒小提。
(6)PCR鉴定:用sgRNA+cas9验证引物对小提质粒进行PCR,产物进行跑1%琼脂糖凝胶电泳,用D2000 marker标定。P出200bp左右片段即为所需片段(如图3),可初步鉴定载体系统。
sgRNA+cas9验证引物-F(SEQ ID NO:5):5'- ATGGTTTCCCATGATTCCTT- 3'
sgRNA+cas9验证引物-R(SEQ ID NO:6):5'-CGACTCGGTGCCACTTTT- 3';
(7)测序鉴定:PCR鉴定成功的质粒生工公司测序;并命名PENTY-U6-EF1a-Cas9-sg1、PENTY-U6-EF1a-Cas9-sg2。
实施例2:敲除效率验证
用含10%胎牛血清的1640培养基于5%CO2,37℃恒温培养SK-BR-3细胞;取对数期细胞以5×105/孔接种到六孔板培养,24h后细胞融合度达到70%-80%时,用Lipofectamine®2000试剂将PENTY-U6-EF1a-Cas9-sg1、PENTY-U6-EF1a-Cas9-sg2各2.5μg分别转染到不同孔中,1个孔只加入转染试剂作为control,转染48小时,消化收集各孔细胞。
收集细胞株提取蛋白质,进行Western blotting方法检测PI3K蛋白在各组细胞中的表达情况;结果显示,敲除PIK3CA的乳腺癌上皮细胞SK-BR-3-sg1、SK-BR-3-sg2实验组对比SK-BR-3-WT中PI3K蛋白表达水平显著降低(图4)。
则说明所提供的sgRNA1、sgRNA2序列所构建出的cas9系统可以有效的降低PI3K的表达量,可以应用于细胞,具体可用于乳腺癌细胞中。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
序列表
<110> 昆明理工大学
<120> 靶向敲除PIK3CA基因的sgRNA及应用
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213> 人工序列(Artificial)
<400> 1
tccgcggctc taaccgcatc ggg 23
<210> 2
<211> 23
<212> DNA
<213> 人工序列(Artificial)
<400> 2
acccgatgcg gttagagccg cgg 23
<210> 3
<211> 29
<212> DNA
<213> 人工序列(Artificial)
<400> 3
gtatttcgat ttcttggctt tatatatct 29
<210> 4
<211> 27
<212> DNA
<213> 人工序列(Artificial)
<400> 4
gttgataacg gactagcctt attttac 27
<210> 5
<211> 20
<212> DNA
<213> 人工序列(Artificial)
<400> 5
atggtttccc atgattcctt 20
<210> 6
<211> 18
<212> DNA
<213> 人工序列(Artificial)
<400> 6
cgactcggtg ccactttt 18
Claims (7)
1.一种靶向敲除PIK3CA基因的sgRNA,其具有下列核苷酸序列之一:
a、sgRNA1:TCCGCGGCTCTAACCGCATCGGG;
b、sgRNA2:ACCCGATGCGGTTAGAGCCGCGG。
2.一种靶向敲除PIK3CA基因表达的CRISPR-Cas9系统,其特征在于:含有权利要求1所述的靶向敲除PIK3CA基因表达的sgRNA的DNA序列。
3.权利要求2所述的靶向敲除PIK3CA基因表达的CRISPR-Cas9系统的构建方法,其特征在于包括如下步骤:
(1)使用XmaⅠ和PmeⅠ内切酶双酶切载体PENTY-U6-EF1a-Cas9,胶回收得到酶切后的CRISPR-Cas9载体;
(2)将靶向敲除PIK3CA基因表达的sgRNA的DNA序列用CRISPR-F、CRISPR-R引物扩增后,再与酶切后的CRISPR-Cas9载体连接,得到靶向PIK3CA基因的CRISPR-Cas9载体系统;
CRISPR-F:5’-GTATTTCGATTTCTTGGCTTTATATATCT-3’;
CRISPR-R:5’-GTTGATAACGGACTAGCCTTATTTTAC-3’。
4.权利要求2所述的靶向敲除PIK3CA基因表达的CRISPR-Cas9系统在制备敲除PIK3CA基因的细胞株中的应用,其特征在于:将靶向敲除PIK3CA基因的CRISPR-Cas9系统转染至目的细胞株中。
5.根据权利要求4所述的应用,其特征在于:目的细胞株为肿瘤细胞株。
6.根据权利要求5所述的应用,其特征在于:肿瘤细胞株为乳腺癌细胞株。
7.根据权利要求6所述的应用,其特征在于:乳腺癌细胞株为人乳腺上皮细胞SK-BR-3。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201710997196.XA CN107828786A (zh) | 2017-10-24 | 2017-10-24 | 靶向敲除PIK3CA基因的sgRNA及应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201710997196.XA CN107828786A (zh) | 2017-10-24 | 2017-10-24 | 靶向敲除PIK3CA基因的sgRNA及应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN107828786A true CN107828786A (zh) | 2018-03-23 |
Family
ID=61648817
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201710997196.XA Pending CN107828786A (zh) | 2017-10-24 | 2017-10-24 | 靶向敲除PIK3CA基因的sgRNA及应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN107828786A (zh) |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102453765A (zh) * | 2011-11-03 | 2012-05-16 | 厦门艾德生物医药科技有限公司 | Pik3ca基因驱动突变的检测探针、引物及试剂盒 |
CN104010494A (zh) * | 2011-07-28 | 2014-08-27 | 霍夫曼-拉罗奇有限公司 | Pik3ca h1047r敲入非人动物乳腺癌模型 |
CN105238799A (zh) * | 2015-10-19 | 2016-01-13 | 昆明理工大学 | 一种乳腺癌pik3ca突变基因及其应用 |
WO2016049024A2 (en) * | 2014-09-24 | 2016-03-31 | The Broad Institute Inc. | Delivery, use and therapeutic applications of the crispr-cas systems and compositions for modeling competition of multiple cancer mutations in vivo |
CN105969875A (zh) * | 2016-06-15 | 2016-09-28 | 昆明理工大学 | 用于检测微量组织中pik3ca基因突变的引物组合及其应用 |
-
2017
- 2017-10-24 CN CN201710997196.XA patent/CN107828786A/zh active Pending
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104010494A (zh) * | 2011-07-28 | 2014-08-27 | 霍夫曼-拉罗奇有限公司 | Pik3ca h1047r敲入非人动物乳腺癌模型 |
CN102453765A (zh) * | 2011-11-03 | 2012-05-16 | 厦门艾德生物医药科技有限公司 | Pik3ca基因驱动突变的检测探针、引物及试剂盒 |
WO2016049024A2 (en) * | 2014-09-24 | 2016-03-31 | The Broad Institute Inc. | Delivery, use and therapeutic applications of the crispr-cas systems and compositions for modeling competition of multiple cancer mutations in vivo |
CN105238799A (zh) * | 2015-10-19 | 2016-01-13 | 昆明理工大学 | 一种乳腺癌pik3ca突变基因及其应用 |
CN105969875A (zh) * | 2016-06-15 | 2016-09-28 | 昆明理工大学 | 用于检测微量组织中pik3ca基因突变的引物组合及其应用 |
Non-Patent Citations (7)
Title |
---|
ELIZABETH IORNS等: "Integrated Functional, Gene Expression and Genomic Analysis for the Identification of Cancer Targets", 《PLOS ONE》 * |
ROBERT J CROWDER等: "PIK3CA and PIK3CB Inhibition Produce Synthetic Lethality When Combined With Estrogen Deprivation in Estrogen Receptor-Positive Breast Cancer", 《CANCER RESEARCH》 * |
WENLI CUI等: "PIK3CACA expression in diffuse large B cell lymphoma tissue and the effect of its knockdown in vitro", 《ONCOTARGETS AND THERAPY》 * |
WHITNEY S HENRY等: "LINC00520 Is Induced by Src, STAT3, and PI3K and Plays a Functional Role in Breast Cancer", 《ONCOTARGET》 * |
于声等: "靶向剪切AAVS1位点CRISPR/Cas9腺病毒系统的构建", 《基因组学与应用生物学》 * |
李凤等: "应用CRISPR/Cas9技术构建MAVS基因敲除的ZR-751乳腺癌细胞株", 《军事医学》 * |
项华丽等: "PIK3CA 基因可成为乳腺癌的一个理想靶向治疗位点", 《中国妇幼保健》 * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN105112445B (zh) | 一种基于CRISPR-Cas9基因敲除技术的miR-205基因敲除试剂盒 | |
CN106566838B (zh) | 一种基于CRISPR-Cas9技术的miR-126全长基因敲除试剂盒及其应用 | |
CN106047877B (zh) | 一种靶向敲除FTO基因的sgRNA及CRISPR/Cas9慢病毒系统与应用 | |
CN108753772A (zh) | 基于CRISPR/Cas技术敲除CAPNS1基因的人神经母细胞瘤细胞系的构建方法 | |
Miyagishima et al. | Two types of FtsZ proteins in mitochondria and red-lineage chloroplasts: the duplication of FtsZ is implicated in endosymbiosis | |
Banerjee et al. | Microalgal bioengineering for sustainable energy development: recent transgenesis and metabolic engineering strategies | |
CN109207515A (zh) | 一种设计和构建猪全基因组CRISPR/Cas9敲除文库的方法 | |
Zou et al. | Improving the efficiency of prime editing with epegRNAs and high-temperature treatment in rice | |
Nicolia et al. | Tomato protoplasts as cell target for ribonucleoprotein (RNP)-mediated multiplexed genome editing | |
CN104818334B (zh) | 与肺腺癌转移相关的微小rna | |
CN107267516B (zh) | 双sgRNA介导的基因精确修饰方法及应用 | |
Kim et al. | A robust and practical CRISPR/crRNA screening system for soybean cultivar editing using LbCpf1 ribonucleoproteins | |
CN110343724A (zh) | 用于筛选和鉴定功能性lncRNA的方法 | |
CN107636155A (zh) | 红冬孢酵母属和红酵母属的d‑氨基酸诱导型基因表达系统 | |
CN106967716A (zh) | 双gRNA、双gRNA文库、双gRNA载体文库及其制备方法和应用 | |
Li et al. | Sequence and insertion sites of murine melanoma-associated retrovirus | |
CN109706148A (zh) | 一种用于敲除BCL11A基因或者BCL11A基因增强子的gRNA、gRNA组合物以及电转方法 | |
Shan et al. | The complete mitochondrial genome of Amorphophallus albus and development of molecular markers for five Amorphophallus species based on mitochondrial DNA | |
CN105861551B (zh) | 联合表达microRNAs抑制乳腺癌细胞增殖的载体及其构建方法和应用 | |
CN103266120A (zh) | 鉴定miRNA靶点的双荧光报告基因载体及其制备方法和应用 | |
CN109837301A (zh) | 人源化幽门螺旋杆菌cagA真核表达载体构建方法 | |
Burnett et al. | Examination of the cell cycle dependence of cytosine and adenine base editors | |
CN109487005A (zh) | 用于扩增山羊地方性鼻内肿瘤病毒全基因组序列的引物 | |
CN109609551A (zh) | 一种利用CRISPR/Cas9+AAV制备通用型CAR-T细胞的方法 | |
CN107828786A (zh) | 靶向敲除PIK3CA基因的sgRNA及应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
RJ01 | Rejection of invention patent application after publication | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180323 |