CN105567692B - 一种特异性靶向QKI基因的sgRNA及其应用 - Google Patents
一种特异性靶向QKI基因的sgRNA及其应用 Download PDFInfo
- Publication number
- CN105567692B CN105567692B CN201610108864.4A CN201610108864A CN105567692B CN 105567692 B CN105567692 B CN 105567692B CN 201610108864 A CN201610108864 A CN 201610108864A CN 105567692 B CN105567692 B CN 105567692B
- Authority
- CN
- China
- Prior art keywords
- qki
- sgrna
- genes
- gene
- seq
- 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.)
- Expired - Fee Related
Links
- 108091027544 Subgenomic mRNA Proteins 0.000 title claims abstract description 43
- 108091034117 Oligonucleotide Proteins 0.000 abstract description 16
- 102100038669 Protein quaking Human genes 0.000 abstract description 12
- 238000000034 method Methods 0.000 abstract description 12
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 abstract description 11
- 238000003209 gene knockout Methods 0.000 abstract description 10
- 230000008685 targeting Effects 0.000 abstract description 10
- 238000001890 transfection Methods 0.000 abstract description 8
- 101150079057 QKI gene Proteins 0.000 abstract description 6
- 239000000969 carrier Substances 0.000 abstract description 5
- 230000008827 biological function Effects 0.000 abstract description 3
- 238000011813 knockout mouse model Methods 0.000 abstract description 2
- 108090000623 proteins and genes Proteins 0.000 description 11
- 238000010356 CRISPR-Cas9 genome editing Methods 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 6
- 239000013612 plasmid Substances 0.000 description 6
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 6
- 108020004414 DNA Proteins 0.000 description 5
- 239000002253 acid Substances 0.000 description 5
- 231100000221 frame shift mutation induction Toxicity 0.000 description 5
- 230000037433 frameshift Effects 0.000 description 5
- 238000011081 inoculation Methods 0.000 description 5
- 238000012163 sequencing technique Methods 0.000 description 5
- 238000000137 annealing Methods 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 230000003321 amplification Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000004925 denaturation Methods 0.000 description 3
- 230000036425 denaturation Effects 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 229950010131 puromycin Drugs 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 108091033409 CRISPR Proteins 0.000 description 2
- 108020004459 Small interfering RNA Proteins 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000010494 dissociation reaction Methods 0.000 description 2
- 230000005593 dissociations Effects 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000010362 genome editing Methods 0.000 description 2
- 239000002777 nucleoside Substances 0.000 description 2
- 125000003835 nucleoside group Chemical group 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000003757 reverse transcription PCR Methods 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 239000012096 transfection reagent Substances 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 101710159080 Aconitate hydratase A Proteins 0.000 description 1
- 101710159078 Aconitate hydratase B Proteins 0.000 description 1
- 108020005544 Antisense RNA Proteins 0.000 description 1
- 238000010354 CRISPR gene editing Methods 0.000 description 1
- 230000007018 DNA scission Effects 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 108020005004 Guide RNA Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 102000044126 RNA-Binding Proteins Human genes 0.000 description 1
- 101710105008 RNA-binding protein Proteins 0.000 description 1
- 241000218636 Thuja Species 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000005782 double-strand break Effects 0.000 description 1
- 230000011559 double-strand break repair via nonhomologous end joining Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000005755 formation reaction Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 210000001320 hippocampus Anatomy 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000006780 non-homologous end joining Effects 0.000 description 1
- 238000011330 nucleic acid test Methods 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 210000002442 prefrontal cortex Anatomy 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 229920002477 rna polymer Polymers 0.000 description 1
- 201000000980 schizophrenia Diseases 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000005406 washing 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
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- 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
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal 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
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
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)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明提供制备特异性靶向目标基因QKI的sgRNA,其序列如SEQ ID NO.1所示。利用本发明提供的sgRNA,合成双链sgRNA寡聚核苷酸,将sgRNA构建至pSpCas9(BB)‑2A‑Puro载体,鉴定并扩增,转染HEK293T细胞获得QKI基因敲除细胞。本发明能够在较短时间内精确靶向人QKI基因并且实现基因敲除,免去了购买QKI基因敲除小鼠分离原代细胞的复杂操作,且价格低廉,有助于进一步深入研究QKI的生物学功能。本发明方法步骤简单、sgRNA靶向性好,QKI基因敲除效率高。
Description
技术领域
本发明属于基因工程领域,更具体地说涉及一种特异性靶向QKI基因的sgRNA,以及CRISPR-Cas9特异性敲除人HEK293T细胞QKI基因的方法。
背景技术
规律成簇间隔短回文重复系统(clustered regularly interspaced shortpalindromic repeat;CRISPR-associated,CRISPR-Cas9)是一种具有核酸内切酶活性的复合体,识别特定的DNA序列,进行特定位点切割造成双链DNA断裂(Double-strand breaks,DSB),在没有模板的条件下,发生非同源重组末端连接(Non-homologous end joining,NHEJ),造成移码突变(frameshift mutation),导致基因敲除。
Cas9靶向切割DNA是通过向导核糖核酸(guide RNA,sgRNA)和靶序列互补识别的原理实现的。sgRNA的特异性决定了基因编辑的精确程度。因此,设计、制备出精确性和特异性靶向目标基因的sgRNA成为CRISPR-Cas9基因敲除的关键技术。
QKI是一类RNA结合蛋白,具有调控RNA剪切、运输等多个生物学功能。
CRISPR-Cas9快速、简便、高效、特异性靶向敲除基因,通过靶向敲除QKI基因,有助于深入研究其功能。而能否设计、制备出精确性和特异性靶向QKI基因的sgRNA以及通过分子生物学方法制备出靶向QKI基因的CRISPR-Cas9系统成为实现QKI基因敲除的关键技术。
参考文献:
Ran,F.A.et al.,Genome engineering using the CRISPR-Cas9system.NATPROTOC 8 2281(2013)。
Lauriat,T.L.et al.,Developmental expression profile of quaking,acandidate gene for schizophrenia,and its target genes in human prefrontalcortex and hippocampus shows regional specificity.J NEUROSCI RES 86 785(2008)。
发明内容
本发明的目的是提供制备特异性靶向目标基因QKI的sgRNA,序列如SEQ ID NO.1所示。
本发明的另一个目的是提供CRISPR-Cas9特异性敲除人HEK293T细胞QKI基因的方法,通过以下步骤实现。
一、sgRNA寡核苷酸的设计和选择
靶向QKI基因的sgRNA的设计按如下原则:
1.在QKI基因上选择特征为5’-N(19)GG或5’-N(20)GG-3’或5’-N(21)GG-3’的序列。
2.sgRNA在QKI基因上的靶向位点位于不同的各种剪切形式的共有外显子上。
3.sgRNA在QKI基因上的靶向位点位于整个基因的前半段,尤其宜在基因的功能结构域中;
按以上原则,设计得到sgRNA,序列为SEQ No.1。
二、合成双链sgRNA寡聚核苷酸
根据选择的sgRNA,去除3’端NGG三个碱基,在其5’端加上CACCG得到正向寡核苷酸;根据选择的sgRNA,去除3’端NGG三个碱基,获得对应DNA的互补链,并且在其5’加上AAAC,3’端加上C得到反向寡核苷酸。上述正向、反向寡核苷酸序列见SEQ NO.2和SEQ NO.3。
分别合成上述正向寡核苷酸和反向寡核苷酸,将合成的sgRNA寡聚核苷酸变性、退火,形成可以连入pSpCas9(BB)真核表达载体的双链。
三、将sgRNA构建至pSpCas9(BB)-2A-Puro载体,鉴定并扩增
1.将退火的sgRNA寡聚核苷酸双链与pSpCas9(BB)-2A-Puro载体(序列见SEQNO.4)连接获得pSpCas9(BB)-2A-Puro-QKIsg质粒(序列见SEQ NO.5)。
2.转化并涂Amp+平板。
3.用SEQ ID NO.6的通用引物U6测序的方法鉴定阳性克隆。
4.37℃摇床摇菌过夜并抽提pSpCas9(BB)-2A-Puro-QKIsg质粒。
四、转染HEK293T细胞获得QKI基因敲除细胞
1、按照DNA&siRNA Transfection Reagent(Polyplus transfection,114-01)的操作手册,将pSpCas9(BB)-2A-Puro-QKIsg质粒转染至HEK293T细胞。
2.转染后细胞加入10μg/ml Puromycin(Merck,540411)药筛,消化存活细胞,将单个细胞接种于96孔板。
3、待细胞扩增后,提取基因组DNA,RT-PCR扩增靶向QKI区域片段,所用引物序列见SEQ NO.7和SEQ NO.8,用Sanger法测序检测sgRNA靶向区域附近基因序列,测序引物见SEQNO.8,确认是否发生移码突变。用Western Blot检测QKI基因已经被敲除。
本发明可以在较短时间内实现QKI基因敲除,免去了购买QKI基因敲除小鼠分离原代细胞的复杂操作,且价格低廉,有助于进一步深入研究QKI的生物学功能。利用本发明制备的特异性靶向人QKI基因的sgRNA能够精确靶向人QKI基因并且实现基因敲除。该制备方法步骤简单、sgRNA靶向性好,QKI基因敲除效率高。
附图说明
Sanger法测序确认QKI阅读框发生移码突变,与参考序列相比缺失22个碱基(图1和图2)。
Western Blot确认QKI基因敲除(图3)。
具体实施方式
下面结合附图和具体的实施例对本发明的技术方案做进一步介绍。
实施例1靶向人QKI基因的sgRNA的设计和合成
1.靶向人QKI基因的sgRNA的设计。
(1)在QKI基因上选择特征为5’-N(19)GG或5’-N(20)GG-3’或5’-N(21)GG-3’的序列。
(2)sgRNA在QKI基因上的靶向位点位于不同的各种剪切形式的共有外显子上。
(3)sgRNA在QKI基因上的靶向位点位于整个基因的前半段,尤其宜在基因的功能结构域中。
按以上原则,设计得到sgRNA,序列为SEQ No.1。
3.靶向人QKI基因的sgRNA寡聚核苷酸的合成和构建。
根据选择的sgRNA,去除3’端NGG三个碱基,在其5’端加上CACCG得到正向寡核苷酸;根据选择的sgRNA,去除3’端NGG三个碱基,获得对应DNA的互补链,并且在其5’加上AAAC,3’端加上C得到反向寡核苷酸。序列见SEQ NO.2和SEQ NO.3。分别合成上述正向寡核苷酸和反向寡核苷酸,将合成的sgRNA寡聚核苷酸变性、退火,形成可以连入pSpCas9(BB)-2A-Puro真核表达载体的双链sgRNA寡聚核苷酸。
变性、退火反应体系为:
1μl正向寡核苷酸(100μM)
1μl反向寡核苷酸(100μM)
1μl T4ligation buffer(New England BioLabs公司,货号:B0202S)
1μl T4PNK(New England BioLabs公司,货号:M0201S)
6μl灭菌水。
在PCR仪中按照以下程序运行:37℃,30min;95℃,5min;0.1℃/sec由95℃降至25℃。
实施例2将双链sgRNA构建至pSpCas9(BB)-2A-Puro载体
将变性、退火后所得双链sgRNA按1:200比例稀释。
将双链sgRNA构建至pSpCas9(BB)-2A-Puro载体(序列见SEQ NO.4),反应体系如下:
100ng pSpCas9(BB)-2A-Puro质粒
2μl稀释后双链sgRNA
2μl Tango buffer,10X(Thermo Scientific,货号BY5)
1μl DTT,10mM(Thermo Scientific,货号R0862)
1μl ATP,10mM(Thermo Scientific,货号AM8110G)
1μl FastDigest BbsⅠ(Thermo Scientific,货号FD1014)
0.5μl T7ligase(Enzymatics,货号L602L)
灭菌水,补足至20μl。
反应条件:37℃5min,21℃5min。共6个循环.
将上述步骤获得的连接产物转化Stbl3感受态细胞(Thermo Scientific,货号C7373-03)并涂Amp+平板(50μg/ml),并挑取克隆。
用如序列表SEQ ID NO.6所示的通用引物U6,用Sanger法测序的方法鉴定获得阳性克隆。
37℃摇床摇菌过夜培养阳性克隆,抽提质粒,获得pSpCas9(BB)-2A-Puro-QKIsg质粒(如序列表SEQ ID NO.5所示)。
实施例3构建人HEK293T细胞QKI基因敲除
1、细胞培养与转染。
(1)HEK293T细胞接种培养于DMEM高糖培养液中(Thermo Scientific,货号11995-065),其中含10%FBS(Thermo Scientific,货号10099-141),penicillin(100U/ml)和streptomycin(100μg/ml)。
(2)在转染前将细胞接种至6孔板中,待70%~80%密度时进行转染。
(3)按照DNA&siRNA Transfection Reagent(Polyplus transfection,114-01)操作手册,将2μg pSpCas9(BB)-2A-Puro-QKIsg质粒转染至每孔细胞中,6~8小时后换液,并加入10μg/ml Puromycin(Merck,540411)药筛,48小时后收取细胞。
2、挑选单克隆:
将Puromycin筛选后细胞消化后计数,取60个细胞接种至一块96孔板上,接种24h后,显微镜下观察,挑选只含单个细胞的孔,继续培养至90%密度。
将上述细胞消化后接种至24孔板,扩大培养,继续培养至90%密度。
3、Sanger法测序鉴定基因突变:
消化细胞,离心后取一半细胞继续培养,一半细胞提取基因组DNA(生工,货号B518401),RT-PCR扩增包含sgRNA靶向QKI的片段,所用引物见SEQ NO.7和SEQ NO.8。
Sanger法测序,测序引物即SEQ NO.8,并与Ensemble数据库参考序列比对,鉴定QKI阅读框有无发生移码突变,结果见图1和图2。
4、Western Blot鉴定QKI敲除情况:
提取对照组和敲除组细胞蛋白,进行蛋白电泳,随后转膜,封闭,加入抗QKI一抗孵育(Abcam,货号ab126742)过夜,次日去除一抗并洗涤30min,加入二抗(Abcam,ab6721)孵育1小时,随后再次洗涤30min后显影,结果见图3。
Claims (1)
1.一种制备特异性靶向目标基因QKI的sgRNA,其特征在于,其序列如SEQ ID NO.1所示。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201610108864.4A CN105567692B (zh) | 2016-02-26 | 2016-02-26 | 一种特异性靶向QKI基因的sgRNA及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201610108864.4A CN105567692B (zh) | 2016-02-26 | 2016-02-26 | 一种特异性靶向QKI基因的sgRNA及其应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN105567692A CN105567692A (zh) | 2016-05-11 |
CN105567692B true CN105567692B (zh) | 2018-08-14 |
Family
ID=55878320
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201610108864.4A Expired - Fee Related CN105567692B (zh) | 2016-02-26 | 2016-02-26 | 一种特异性靶向QKI基因的sgRNA及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN105567692B (zh) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106636199A (zh) * | 2016-12-02 | 2017-05-10 | 中国人民解放军军事医学科学院野战输血研究所 | 用CRISPR/Cas9技术易于筛选获得目的基因敲除细胞系的方法及产品 |
CN106987560B (zh) * | 2017-03-29 | 2020-12-08 | 中国农业科学院上海兽医研究所 | Rk-13细胞hbb基因敲除稳定株的构建方法 |
CN108913692B (zh) * | 2018-07-17 | 2019-07-09 | 浙江大学 | 特异性靶向SATB1基因的sgRNA及其在转录激活中的应用 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102416182A (zh) * | 2011-12-06 | 2012-04-18 | 厦门大学 | miR-574-5p的用途 |
CN103668472A (zh) * | 2013-12-31 | 2014-03-26 | 北京大学 | 利用CRISPR/Cas9系统构建真核基因敲除文库的方法 |
-
2016
- 2016-02-26 CN CN201610108864.4A patent/CN105567692B/zh not_active Expired - Fee Related
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102416182A (zh) * | 2011-12-06 | 2012-04-18 | 厦门大学 | miR-574-5p的用途 |
CN103668472A (zh) * | 2013-12-31 | 2014-03-26 | 北京大学 | 利用CRISPR/Cas9系统构建真核基因敲除文库的方法 |
Non-Patent Citations (2)
Title |
---|
Cas9-sgRNA共质粒系统提高在AAVS1位点的打靶效率;俞珺瑶等;《生物技术通讯》;20150731;第26卷(第4期);第449-452页 * |
通过CRISPR/Cas9系统敲除人源PDE10A基因;梁振伟等;《基础医学与临床》;20140430;第34卷(第4期);第439-443页 * |
Also Published As
Publication number | Publication date |
---|---|
CN105567692A (zh) | 2016-05-11 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Kwart et al. | Precise and efficient scarless genome editing in stem cells using CORRECT | |
CN107435051B (zh) | 一种通过CRISPR/Cas9系统快速获得大片段缺失的细胞系基因敲除方法 | |
US20220033858A1 (en) | Crispr oligoncleotides and gene editing | |
US20210340566A1 (en) | Compositions and methods for differential cas9 gene labeling and/or editing | |
Liu et al. | Comprehensive characterization of distinct states of human naive pluripotency generated by reprogramming | |
Cheng et al. | Single-cell RNA-seq reveals cellular heterogeneity of pluripotency transition and X chromosome dynamics during early mouse development | |
Ran et al. | Genome engineering using the CRISPR-Cas9 system | |
Weinberg et al. | Reconstitution of simian virus 40 DNA replication with purified proteins. | |
Grobarczyk et al. | Generation of isogenic human iPS cell line precisely corrected by genome editing using the CRISPR/Cas9 system | |
Liang et al. | Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection | |
Torres-Ruiz et al. | Efficient recreation of t (11; 22) EWSR1-FLI1+ in human stem cells using CRISPR/Cas9 | |
Yumlu et al. | Gene editing and clonal isolation of human induced pluripotent stem cells using CRISPR/Cas9 | |
CN105567692B (zh) | 一种特异性靶向QKI基因的sgRNA及其应用 | |
US10316288B2 (en) | Neural regenerating cells with alterations in DNA methylation | |
Maguire et al. | Highly efficient CRISPR‐Cas9‐mediated genome editing in human pluripotent stem cells | |
KR20220070443A (ko) | 반복성 dna와 연관된 장애의 치료용 조성물 및 방법 | |
US20220259646A1 (en) | Compositions and methods of labeling nucleic acids and sequencing and analysis thereof | |
Chateauvieux et al. | Molecular profile of mouse stromal mesenchymal stem cells | |
Valton et al. | Efficient strategies for TALEN-mediated genome editing in mammalian cell lines | |
Sargent et al. | Nuclease-mediated double-strand break (DSB) enhancement of small fragment homologous recombination (SFHR) gene modification in human-induced pluripotent stem cells (hiPSCs) | |
Smith et al. | Locus-specific DNA methylation editing in melanoma cell lines using a CRISPR-based system | |
Petazzi et al. | CRISPR/Cas9–mediated gene knockout and knockin human iPSCs | |
Liu et al. | Highly precise base editing with CC context-specificity using engineered human APOBEC3G-nCas9 fusions | |
Zeller et al. | Hierarchical chromatin regulation during blood formation uncovered by single-cell sortChIC | |
Carlson-Stevermer et al. | Genome editing in human pluripotent stem cells |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant | ||
CF01 | Termination of patent right due to non-payment of annual fee | ||
CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20180814 Termination date: 20210226 |