WO2020000457A1 - Grna targeting sequence that specifically targets human kat13d gene and use thereof - Google Patents

Grna targeting sequence that specifically targets human kat13d gene and use thereof Download PDF

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WO2020000457A1
WO2020000457A1 PCT/CN2018/093867 CN2018093867W WO2020000457A1 WO 2020000457 A1 WO2020000457 A1 WO 2020000457A1 CN 2018093867 W CN2018093867 W CN 2018093867W WO 2020000457 A1 WO2020000457 A1 WO 2020000457A1
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kat13d
gene
grna
human
vector
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PCT/CN2018/093867
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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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Publication of WO2020000457A1 publication Critical patent/WO2020000457A1/en

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    • 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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Definitions

  • the invention belongs to the technical field of gene editing, and particularly relates to a gRNA guide sequence that specifically targets a human KAT13D gene and an application thereof.
  • Circadian rhythm is a kind of biological rhythm. It is a rhythm that appears under the combined effect of exogenous and endogenous factors. Its endogenous factor is a closed-loop oscillatory system composed of a set of biorhythmic genes and their gene products.
  • Biorhythm genes include KAT13D, Per1, Per2, Per3, Cry1, Cry2, BMAL1, Timless, etc., which play a key role in maintaining the body's body temperature, breathing, sleep, eating, blood pressure, blood glucose, endocrine and many other homeostasis.
  • KAT13D gene plays a central role in this system. Therefore, the study of the CLOCK gene is very important, but the lack of means for targeted knock-out of KAT13D gene expression in the prior art has caused certain obstacles to the progress of related research.
  • the present invention provides a gRNA targeting sequence that specifically targets the human KAT13D gene.
  • the gRNA targeting sequence can be used to knock out the human KAT13D gene, thereby suppressing or eliminating the expression of KAT13D.
  • Yet another object of the present invention is to provide an application of the gRNA-directed sequence specifically targeting the human KAT13D gene.
  • a gRNA targeting sequence that specifically targets the human KAT13D gene is KAT13D-gRNA, and its nucleotide sequence is:
  • KAT13D-gRNA 5’- CAAACGCCAGCGGCGGTGAC -3 ’;
  • a method for knocking out the human KAT13D gene using the CRISPR / Cas9 system includes the following steps:
  • step b ligating the double strand prepared in step a with the Cas9 vector to obtain a recombinant knockout expression vector
  • the recombinant knockout expression vector prepared in step b is transfected into the target cells, and puromycin is selected to obtain cells that have successfully knocked out the KAT13D gene.
  • the Cas9 vector in step b is a px459 vector
  • the target cell in step c is a C6 cell
  • the concentration of puromycin in step c is 1.0 ⁇ g / ml.
  • the present invention has the following advantages and effects:
  • the present invention designs and synthesizes two single-stranded oligo sequences according to the gRNA-directed sequence, anneals to form a double-strand, and then ligates with the Cas9 vector.
  • the Cas9 vector is used to introduce the gRNA and CRISPR system into the target cell.
  • the Cas9 protein will be found under the guidance of the gRNA
  • the matching DNA sequence is cut to realize the KAT13D gene knockout.
  • the vector contains a puromycin resistance gene. Puromycin can be used to screen cells, and cells that have not been transferred into the vector can be screened out.
  • Figure 1 shows the Western Blot results of C6 cells in the control and experimental groups.
  • Example 1 Targeting KAT13D genetic gRNA design
  • a gRNA targeting human KAT13D gene was designed.
  • the 20nt oligonucleotide gRNA targeting sequence is: KAT13D-gRNA: 5'- CAAACGCCAGCGGCGGTGAC -3 ', then add CACC to its 5' end to obtain a forward oligonucleotide, and 5 'to its reverse complementary sequence Add AAAC to the end.
  • the above-mentioned forward oligonucleotide and reverse oligonucleotide were synthesized separately, denatured at 95 ° C, and annealed to form a double-stranded DNA molecule that can be ligated into the px459 vector.
  • the px459 vector has a Bbs I digestion site, which is digested with Bbs I.
  • the digestion system (20 ⁇ l) is: Bbs I 1 ⁇ L; 10 ⁇ FastDigest buffer 2 ⁇ L; plasmid 1 ⁇ g; ddH2O supplemented to 20 ⁇ l; digestion conditions are: : Digestion for 1 h at 37 ° C. After the digestion is completed, the gel is recovered and purified.
  • the digested vector px459 and the annealed double strand obtained in Example 1 were ligated with T4 ligase.
  • the ligation system (10 ⁇ l) was: annealed double strand (KAT13D-gRNA) 2 ⁇ l, px459 vector 2 ⁇ l, 10 ⁇ T4 DNA Ligase Buffer 1 ⁇ l, T4 DNA Ligase 1 ⁇ l, ddH2O to make up to 10 ⁇ l; ligation conditions: ligation at 16 ° C overnight.
  • the ligation product is transformed into competent cells Stbl3.
  • the specific transformation method is: take out the competent cells Stbl3 at -80 ° C, and dissolve them in an ice bath; then take 1 ⁇ l of the above-mentioned ligation products to 50 ⁇ l of competent cells and mix for 30 minutes on ice Do not shake during 42 s water bath for 60 s; cool in ice bath for 2 min; then add 800 ⁇ l LB medium and shake at 37 °C for 30 min; LB plate coated with 100 ⁇ g / ml ampicillin and culture overnight. After picking positive clones Shake at 37 ° C overnight for expansion and send for sequencing. The correct sequencing is the required Cas9 vector targeting KAT13D gene, named px459-KAT13D vector.
  • the correct strain was sequenced and identified in Example 2 and placed in an LB liquid medium having an ampicillin concentration of 100 ⁇ g / ml, and cultured at 250 rpm and 37 ° C. with shaking for 12-16 hours. Collect the bacterial solution by centrifugation at 10,000 rpm at 4 ° C, discard the supernatant, collect the bacterial cells, and then extract the plasmid according to the instructions of the Endo-Free Plasmid Mini Kit kit to obtain the px459-KAT13D vector without endotoxin.
  • Embodiment 4 C6 Transfection of cells
  • C6 cells should be cultured until the fusion rate of C6 cells reaches 50% to 60%.
  • the optimal transfection time is 12 to 18 hours after inoculation. Change the fresh culture medium before transfection. Add 3 ml medium to a 60 mm culture dish. Transfection 4 ⁇ g of px459-KAT13D plasmid was introduced according to the instructions of the Lipofectamine 2000 kit. 48 hours after transfection, 1 ⁇ g / ml puromycin was added for screening for 7 days. After the screening was completed, the concentration of puromycin was reduced to 0.5 ⁇ g / ml and the cells were expanded.
  • Embodiment 5 Western Blot Detection of transfection effect
  • C6 cells without any treatment were used as the control group, and the cells selected in Example 4 were used as the experimental group.
  • 100-200 ⁇ l of 5 ⁇ SDS-PAGE loading buffer was added, and the mixture was boiled in boiling water for 5 minutes. 15 ⁇ l of SDS was loaded.
  • -PAGE protein electrophoresis After electrophoresis, semi-dry transfer with conventional protein, blocking with 10% skim milk powder for 2 h, put the blocked PVDF membrane in rabbit anti-human KAT13D antibody, rinse the buffer 3 times, and then transfer the membrane to goat anti-rabbit secondary antibody Buffer, incubate at room temperature for 60 min, and rinse 4 times with rinsing buffer.
  • the Western blot membrane was developed and detected by ECL, and the results are shown in FIG. 1. It can be seen that the KAT13D protein band cannot be detected by Western Blot in the KAT13D frameshift mutant C6 cells, while the control group has a KAT13D protein band, indicating that the gRNA sequence used for knocking out the KAT13D gene of human cells can achieve KAT13D Gene knockout.
  • the present invention has the following advantages and effects:
  • the present invention designs and synthesizes two single-stranded oligo sequences according to the gRNA-directed sequence, anneals to form a double-strand, and then ligates with the Cas9 vector.
  • the Cas9 vector is used to introduce the gRNA and CRISPR system into the target cell.
  • the Cas9 protein will be found under the guidance of the gRNA.
  • the matching DNA sequence is cut to realize the KAT13D gene knockout.
  • the vector contains a puromycin resistance gene. Puromycin can be used to screen cells, and cells that have not been transferred into the vector can be screened out.

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Abstract

Disclosed are a gRNA targeting sequence that specifically targets the human KAT13D gene and the use thereof, wherein the nucleotide sequence of the gRNA targeting sequence is 5'-CAAACGCCAGCGGCGGTGAC-3'. The knockout of the KAT13D gene is achieved by introducing the gRNA and CRISPR system into target cells using the Cas9 vector.

Description

一种特异靶向人KAT13D基因的gRNA导向序列及其应用GRNA-directed sequence specifically targeting human KAT13D gene and application thereof 技术领域Technical field
本发明属于基因编辑技术领域，特别涉及一种特异靶向人KAT13D基因的gRNA导向序列及其应用。The invention belongs to the technical field of gene editing, and particularly relates to a gRNA guide sequence that specifically targets a human KAT13D gene and an application thereof.
背景技术Background technique
生物节律是指在漫长的生物进化过程中,为了适应自然界周而复始的周期变化,生物体从单细胞到高等动植物以及人类的所有生命活动均形成按照一定规律运行的。昼夜节律是生物节律的一种，它是在外源性因素和内源性因素的共同作用下所呈现出的节律。它的内源性因素是由一组生物节律基因和其基因产物相互作用构成的一个闭环震荡系统。Biological rhythm means that in the long process of biological evolution, in order to adapt to the cyclical changes in nature, all life activities of organisms from single cells to higher plants and animals and humans are formed to operate in accordance with certain laws. Circadian rhythm is a kind of biological rhythm. It is a rhythm that appears under the combined effect of exogenous and endogenous factors. Its endogenous factor is a closed-loop oscillatory system composed of a set of biorhythmic genes and their gene products.
技术问题technical problem
生物节律基因包括KAT13D、Per1、Per2、Per3、Cry1、Cry2、BMAL1、Timless等，在维持机体的体温、呼吸、睡眠、进食、血压、血糖、内分泌等诸多稳态中发挥着关键作用。其中，KAT13D基因在这个系统中起着核心作用，因此，对CLOCK基因的研究十分重要，但现有技术中缺乏靶向敲除KAT13D基因表达的手段，对相关研究的进展造成了一定的阻碍。Biorhythm genes include KAT13D, Per1, Per2, Per3, Cry1, Cry2, BMAL1, Timless, etc., which play a key role in maintaining the body's body temperature, breathing, sleep, eating, blood pressure, blood glucose, endocrine and many other homeostasis. Among them, the KAT13D gene plays a central role in this system. Therefore, the study of the CLOCK gene is very important, but the lack of means for targeted knock-out of KAT13D gene expression in the prior art has caused certain obstacles to the progress of related research.
技术解决方案Technical solutions
针对上述问题，本发明提供一种特异靶向人KAT13D基因的gRNA导向序列，该gRNA导向序列可以用于敲除人KAT13D基因，进而抑制或消除KAT13D的表达。In view of the above problems, the present invention provides a gRNA targeting sequence that specifically targets the human KAT13D gene. The gRNA targeting sequence can be used to knock out the human KAT13D gene, thereby suppressing or eliminating the expression of KAT13D.
本发明的再一目的在于提供上述特异靶向人KAT13D基因的gRNA导向序列的应用。Yet another object of the present invention is to provide an application of the gRNA-directed sequence specifically targeting the human KAT13D gene.
本发明的目的通过下述技术方案实现：The object of the present invention is achieved by the following technical solutions:
一种特异靶向人KAT13D基因的gRNA导向序列，为KAT13D-gRNA，其核苷酸序列为： A gRNA targeting sequence that specifically targets the human KAT13D gene is KAT13D-gRNA, and its nucleotide sequence is:
KAT13D-gRNA：5’- CAAACGCCAGCGGCGGTGAC -3’；KAT13D-gRNA: 5’- CAAACGCCAGCGGCGGTGAC -3 ’;
一种利用CRISPR/Cas9系统敲除人KAT13D基因的方法，包含如下步骤：A method for knocking out the human KAT13D gene using the CRISPR / Cas9 system includes the following steps:
a. 在上述的gRNA导向序列的5 '端加上CACC得到正向寡核苷酸；同时根据导向序列获得其对应的DNA互补链，并且在其5 '端加上AAAC得到反向寡核苷酸；分别合成上述正向寡核苷酸和反向寡核苷酸，将合成的正向寡核苷酸和反向寡核苷酸变性，退火，形成双链；a. Adding CACC to the 5 'end of the above gRNA guide sequence to obtain a forward oligonucleotide; meanwhile, to obtain the corresponding complementary DNA strand according to the guide sequence, and adding AAAC to the reverse oligonucleotide to obtain a reverse oligonucleoside. Acid; synthesize the aforementioned forward oligonucleotide and reverse oligonucleotide, respectively, and denature the synthesized forward oligonucleotide and reverse oligonucleotide, and anneal to form a double strand;
b. 将步骤a制得的双链与Cas9载体连接，得到重组敲除表达载体；b. ligating the double strand prepared in step a with the Cas9 vector to obtain a recombinant knockout expression vector;
c. 将步骤b制得的重组敲除表达载体转染至目的细胞中，嘌呤霉素筛选，得到成功敲除KAT13D基因的细胞。c. The recombinant knockout expression vector prepared in step b is transfected into the target cells, and puromycin is selected to obtain cells that have successfully knocked out the KAT13D gene.
进一步的，步骤b中所述Cas9载体为px459载体；Further, the Cas9 vector in step b is a px459 vector;
进一步的，步骤c中所述目的细胞为C6细胞；Further, the target cell in step c is a C6 cell;
进一步的，步骤c中所述嘌呤霉素的浓度为1.0 μg/ml。Further, the concentration of puromycin in step c is 1.0 μg / ml.
有益效果Beneficial effect
本发明相对于现有技术，具有如下的优点及效果：Compared with the prior art, the present invention has the following advantages and effects:
1、本发明根据gRNA导向序列设计合成两条单链oligo序列，退火形成双链，然后与Cas9载体连接，利用Cas9载体将gRNA以及CRISPR系统引入目标细胞中，Cas9蛋白会在gRNA的引导下找到与其匹配的DNA序列，进行剪切，实现KAT13D基因的敲除。1. The present invention designs and synthesizes two single-stranded oligo sequences according to the gRNA-directed sequence, anneals to form a double-strand, and then ligates with the Cas9 vector. The Cas9 vector is used to introduce the gRNA and CRISPR system into the target cell. The Cas9 protein will be found under the guidance of the gRNA The matching DNA sequence is cut to realize the KAT13D gene knockout.
2、载体中含有嘌呤霉素抗性基因，利用嘌呤霉素对细胞进行筛选，可将未转入载体的细胞筛选淘汰。2. The vector contains a puromycin resistance gene. Puromycin can be used to screen cells, and cells that have not been transferred into the vector can be screened out.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1为对照组和实验组C6细胞的Western Blot结果图。Figure 1 shows the Western Blot results of C6 cells in the control and experimental groups.
本发明的实施方式Embodiments of the invention
实施例中所使用的细胞株均购自ATCC，px459载体购自Addgene，内切酶Bbs I购自Thermo，Endo-Free Plasmid Mini Kit购自Omega-biotek，Lipofectamine 2000购自Invitrogen，T4 DNA连接酶购自NEB，嘌呤霉素购自Sigma。All cell lines used in the examples were purchased from ATCC, px459 vector was purchased from Addgene, endonuclease Bbs I was purchased from Thermo, Endo-Free Plasmid Mini Kit was purchased from Omega-biotek, Lipofectamine 2000 was purchased from Invitrogen, T4 DNA ligase Commercially available from NEB and puromycin from Sigma.
实施例一：靶向Example 1: Targeting KAT13DKAT13D 基因的genetic gRNAgRNA 设计design
根据人KAT13D基因的基因组序列，设计1个靶向人KAT13D基因的gRNA。20nt的寡核苷酸gRNA导向序列为：KAT13D-gRNA：5’- CAAACGCCAGCGGCGGTGAC -3’，然后在其5 '端加上CACC得到正向寡核苷酸，并且在其反向互补序列的5 '端加上AAAC。分别合成上述正向寡核苷酸和反向寡核苷酸，95℃变性，退火，形成可以连入px459载体的双链DNA分子。According to the genomic sequence of human KAT13D gene, a gRNA targeting human KAT13D gene was designed. The 20nt oligonucleotide gRNA targeting sequence is: KAT13D-gRNA: 5'- CAAACGCCAGCGGCGGTGAC -3 ', then add CACC to its 5' end to obtain a forward oligonucleotide, and 5 'to its reverse complementary sequence Add AAAC to the end. The above-mentioned forward oligonucleotide and reverse oligonucleotide were synthesized separately, denatured at 95 ° C, and annealed to form a double-stranded DNA molecule that can be ligated into the px459 vector.
实施例二：构建表达Example 2: Constructing expressions gRNAgRNA 的载体Carrier
px459载体有Bbs I酶切位点，用Bbs I酶切，其中酶切体系（20μl）为：Bbs Ⅰ 1μL；10× FastDigest buffer 2 μL；质粒1 μg；ddH2O补足至20 μl ；酶切条件为：37℃酶切1 h,。酶切完成后进行胶回收纯化。The px459 vector has a Bbs I digestion site, which is digested with Bbs I. The digestion system (20 μl) is: Bbs I 1 μL; 10 × FastDigest buffer 2 μL; plasmid 1 μg; ddH2O supplemented to 20 μl; digestion conditions are: : Digestion for 1 h at 37 ° C. After the digestion is completed, the gel is recovered and purified.
将酶切后的载体px459分别与实施例一中获得的退火双链利用T4连接酶进行连接，连接体系（10 μl）为：退火双链(KAT13D-gRNA) 2 μl，px459载体2 μl，10 × T4 DNA Ligase Buffer 1 μl，T4 DNA Ligase 1 μl，ddH2O补足至10 μl；连接条件：16℃连接过夜。The digested vector px459 and the annealed double strand obtained in Example 1 were ligated with T4 ligase. The ligation system (10 μl) was: annealed double strand (KAT13D-gRNA) 2 μl, px459 vector 2 μl, 10 × T4 DNA Ligase Buffer 1 μl, T4 DNA Ligase 1 μl, ddH2O to make up to 10 μl; ligation conditions: ligation at 16 ° C overnight.
将连接产物转化感受态细胞Stbl3，具体转化方法为：-80℃取出感受态细胞Stbl3，冰浴溶解；然后取50 μl感受态细胞中加入1 μl的上述连接产物，混匀后冰浴30min；42℃水浴60 s，过程中勿摇动；冰浴冷却2 min；然后加入800 μl LB培养基，37℃摇床30min；涂含100 μg/ml氨苄青霉素的LB板培养过夜，挑取阳性克隆后37℃摇床过夜进行扩大培养并送测序。测序正确的即为所需的靶向KAT13D基因的Cas9载体，命名为px459-KAT13D载体。The ligation product is transformed into competent cells Stbl3. The specific transformation method is: take out the competent cells Stbl3 at -80 ° C, and dissolve them in an ice bath; then take 1 μl of the above-mentioned ligation products to 50 μl of competent cells and mix for 30 minutes on ice Do not shake during 42 s water bath for 60 s; cool in ice bath for 2 min; then add 800 μl LB medium and shake at 37 ℃ for 30 min; LB plate coated with 100 μg / ml ampicillin and culture overnight. After picking positive clones Shake at 37 ° C overnight for expansion and send for sequencing. The correct sequencing is the required Cas9 vector targeting KAT13D gene, named px459-KAT13D vector.
实施例三：无内毒素质粒Example 3: Endotoxin-free plasmid DNADNA 的制备Preparation
取实施例二中测序鉴定正确的菌株，置于氨苄青霉素浓度为100 μg/ml的LB液体培养基中，250 rpm、37℃振荡培养12-16 h。4℃，10000 rpm离心收集菌液，弃上清，收集菌体，然后按照Endo-Free Plasmid Mini Kit试剂盒说明书操作步骤提取质粒，得无内毒素的px459-KAT13D载体。The correct strain was sequenced and identified in Example 2 and placed in an LB liquid medium having an ampicillin concentration of 100 μg / ml, and cultured at 250 rpm and 37 ° C. with shaking for 12-16 hours. Collect the bacterial solution by centrifugation at 10,000 rpm at 4 ° C, discard the supernatant, collect the bacterial cells, and then extract the plasmid according to the instructions of the Endo-Free Plasmid Mini Kit kit to obtain the px459-KAT13D vector without endotoxin.
实施例四：Embodiment 4: C6C6 细胞的转染Transfection of cells
培养C6细胞，待C6细胞的融合率达到50％～60％，接种后12～18h为最佳转染时间；转染前更换新鲜培养液，60 mm培养皿中加入3 ml培养基；转染时按照Lipofectamine 2000试剂盒说明书导入4μg的px459-KAT13D质粒，转染后48 h，加入1 μg/ml 嘌呤霉素筛选7 d。筛选完成后，将嘌呤霉素的浓度降为0.5 μg/ml继续扩大培养细胞。C6 cells should be cultured until the fusion rate of C6 cells reaches 50% to 60%. The optimal transfection time is 12 to 18 hours after inoculation. Change the fresh culture medium before transfection. Add 3 ml medium to a 60 mm culture dish. Transfection 4 μg of px459-KAT13D plasmid was introduced according to the instructions of the Lipofectamine 2000 kit. 48 hours after transfection, 1 μg / ml puromycin was added for screening for 7 days. After the screening was completed, the concentration of puromycin was reduced to 0.5 μg / ml and the cells were expanded.
实施例五：Embodiment 5: Western BlotWestern Blot 检测转染效果Detection of transfection effect
以未经任何处理的C6细胞作为对照组，实施例四中筛选出的细胞为实验组，分别加入100-200μl 5 × SDS-PAGE上样缓冲液，沸水煮5 min，取15 μl 上样SDS-PAGE 蛋白电泳。电泳完毕后，按照常规蛋白半干转，10%脱脂奶粉封闭2 h，将封闭后的PVDF膜置于兔抗人KAT13D抗体，缓冲液漂洗3次后，再将膜转移至山羊抗兔二抗缓冲液，室温孵育60 min，再用漂洗缓冲漂洗4次。漂洗完毕后将蛋白印迹膜用ECL显影检测，结果如图1所示。可以看到，KAT13D移码基因突变C6细胞中Western Blot检测不到KAT13D蛋白条带，而对照组则有KAT13D蛋白条带出现，说明所述用于敲除人细胞KAT13D基因的gRNA序列可以实现KAT13D基因的敲除。C6 cells without any treatment were used as the control group, and the cells selected in Example 4 were used as the experimental group. 100-200 μl of 5 × SDS-PAGE loading buffer was added, and the mixture was boiled in boiling water for 5 minutes. 15 μl of SDS was loaded. -PAGE protein electrophoresis. After electrophoresis, semi-dry transfer with conventional protein, blocking with 10% skim milk powder for 2 h, put the blocked PVDF membrane in rabbit anti-human KAT13D antibody, rinse the buffer 3 times, and then transfer the membrane to goat anti-rabbit secondary antibody Buffer, incubate at room temperature for 60 min, and rinse 4 times with rinsing buffer. After the rinsing was completed, the Western blot membrane was developed and detected by ECL, and the results are shown in FIG. 1. It can be seen that the KAT13D protein band cannot be detected by Western Blot in the KAT13D frameshift mutant C6 cells, while the control group has a KAT13D protein band, indicating that the gRNA sequence used for knocking out the KAT13D gene of human cells can achieve KAT13D Gene knockout.
工业实用性Industrial applicability
本发明相对于现有技术，具有如下的优点及效果：Compared with the prior art, the present invention has the following advantages and effects:
1、本发明根据gRNA导向序列设计合成两条单链oligo序列，退火形成双链，然后与Cas9载体连接，利用Cas9载体将gRNA以及CRISPR系统引入目标细胞中，Cas9蛋白会在gRNA的引导下找到与其匹配的DNA序列，进行剪切，实现KAT13D基因的敲除。1. The present invention designs and synthesizes two single-stranded oligo sequences according to the gRNA-directed sequence, anneals to form a double-strand, and then ligates with the Cas9 vector. The Cas9 vector is used to introduce the gRNA and CRISPR system into the target cell. The Cas9 protein will be found under the guidance of the gRNA. The matching DNA sequence is cut to realize the KAT13D gene knockout.
2、载体中含有嘌呤霉素抗性基因，利用嘌呤霉素对细胞进行筛选，可将未转入载体的细胞筛选淘汰。2. The vector contains a puromycin resistance gene. Puromycin can be used to screen cells, and cells that have not been transferred into the vector can be screened out.

Claims (5)

  1. 一种特异靶向人KAT13D基因的gRNA导向序列，其特征在于：所述的特异靶向人KAT13D基因的gRNA导向序列为KAT13D-gRNA，其核苷酸序列为：A gRNA targeting sequence specifically targeting the human KAT13D gene is characterized in that the gRNA targeting sequence specifically targeting the human KAT13D gene is KAT13D-gRNA, and its nucleotide sequence is:
    KAT13D-gRNA：5’- CAAACGCCAGCGGCGGTGAC -3’。KAT13D-gRNA: 5'- CAAACGCCAGCGGCGGTGAC -3 '.
  2. 一种利用CRISPR/Cas9系统敲除人KAT13D基因的方法，其特征在于包含如下步骤：A method for knocking out the human KAT13D gene using the CRISPR / Cas9 system is characterized in that it comprises the following steps:
    a. 在权利要求1所述的gRNA导向序列的5 '端加上CACC得到正向寡核苷酸；同时根据导向序列获得其对应的DNA互补链，并且在其5 '端加上AAAC得到反向寡核苷酸；分别合成上述正向寡核苷酸和反向寡核苷酸，将合成的正向寡核苷酸和反向寡核苷酸变性，退火，形成双链；a. Adding CACC to the 5 ′ end of the gRNA guide sequence according to claim 1 to obtain a forward oligonucleotide; obtaining the corresponding complementary DNA strand according to the guide sequence, and adding AAAC to the 5 ′ end to obtain a reverse oligonucleotide. To the oligonucleotide; synthesize the above-mentioned forward oligonucleotide and reverse oligonucleotide, respectively, and denature the synthesized forward oligonucleotide and reverse oligonucleotide, and anneal to form a double strand;
    b. 将步骤a制得的双链与Cas9载体连接，得到重组敲除表达载体；b. ligating the double strand prepared in step a with the Cas9 vector to obtain a recombinant knockout expression vector;
    c. 将步骤b制得的重组敲除表达载体转染至目的细胞中，嘌呤霉素筛选，得到成功敲除KAT13D基因的细胞。c. The recombinant knockout expression vector prepared in step b is transfected into the target cells, and puromycin is selected to obtain cells that have successfully knocked out the KAT13D gene.
  3. 根据权利要求2所述的利用CRISPR/Cas9系统敲除人KAT13D基因的方法，其特征在于：步骤b中所述Cas9载体为px459载体。The method for knocking out the human KAT13D gene using the CRISPR / Cas9 system according to claim 2, wherein the Cas9 vector in step b is a px459 vector.
  4. 根据权利要求2所述的利用CRISPR/Cas9系统敲除人KAT13D基因的方法，其特征在于：步骤c中所述目的细胞为C6细胞。The method for knocking out the human KAT13D gene using the CRISPR / Cas9 system according to claim 2, wherein the target cell in step c is a C6 cell.
  5. 根据权利要求2所述的利用CRISPR/Cas9系统敲除人KAT13D基因的方法，其特征在于：步骤c中所述嘌呤霉素的浓度为1.0 μg/ml。The method for knocking out the human KAT13D gene using the CRISPR / Cas9 system according to claim 2, wherein the concentration of said puromycin in step c is 1.0 μg / ml.
PCT/CN2018/093867 2018-06-29 2018-06-29 Grna targeting sequence that specifically targets human kat13d gene and use thereof WO2020000457A1 (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101418293A (en) * 2008-12-03 2009-04-29 暨南大学 Tiny RNA-21 antisense oligonucleotides and use thereof
CN105979954A (en) * 2013-11-13 2016-09-28 在配料公司 Treatment or prophylaxis of circadian protein related conditions
CN106434663A (en) * 2016-10-12 2017-02-22 遵义医学院 Method for CRISPR/Cas9 targeted knockout of human ezrin gene enhancer key region and specific gRNA thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101418293A (en) * 2008-12-03 2009-04-29 暨南大学 Tiny RNA-21 antisense oligonucleotides and use thereof
CN105979954A (en) * 2013-11-13 2016-09-28 在配料公司 Treatment or prophylaxis of circadian protein related conditions
CN106434663A (en) * 2016-10-12 2017-02-22 遵义医学院 Method for CRISPR/Cas9 targeted knockout of human ezrin gene enhancer key region and specific gRNA thereof

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