WO2020000454A1 - 一种表达LwCas13a基因的质粒、构建方法及其应用 - Google Patents

一种表达LwCas13a基因的质粒、构建方法及其应用 Download PDF

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WO2020000454A1
WO2020000454A1 PCT/CN2018/093864 CN2018093864W WO2020000454A1 WO 2020000454 A1 WO2020000454 A1 WO 2020000454A1 CN 2018093864 W CN2018093864 W CN 2018093864W WO 2020000454 A1 WO2020000454 A1 WO 2020000454A1
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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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  • the invention belongs to the field of biological engineering and biotechnology, and particularly relates to a plasmid expressing the LwCas13a gene, a construction method and an application thereof.
  • RNA interference refers to the phenomenon that when a double-stranded RNA homologous to an endogenous mRNA coding region is introduced into a cell, the mRNA is degraded to cause gene silencing, which is a phenomenon that inhibits the expression of specific genes in the body.
  • the double-stranded RNA introduced by foreign means or by various methods such as transgene and virus infection will be gradually cleaved into small molecules of 19 ⁇ 23nt by an ATP-dependent manner, Dicer, an enzyme that can specifically recognize double-stranded RNA.
  • Interfering RNA fragments siRNA double-strand is dissociated under the action of RISC, the homologous target RNA is bound, and the target mRNA is cut by the action of endonuclease, thereby blocking gene expression.
  • RNA-targeted CRISPR enzyme Cas13a
  • CRISPR / Cas13a can be used to cut specific RNA sequences in bacterial cells.
  • LwCas13a is generally used in the form of pure protein, and the RNA in eukaryotic cells is cleaved under the guidance of crRNA. There has been no report on the expression of LwCas13a by eukaryotic expression vectors for RNA cutting.
  • a plasmid expressing the LwCas13a gene, pRNAT-LwCas13a-Neo includes the recombinant construction of the CDS sequence of the LwCas13a gene and the pRNAT-U6.1 / Neo eukaryotic expression vector.
  • the nucleotide sequence is shown in SEQ ID ID NO.1.
  • a method for constructing the plasmid expressing the LwCas13a gene includes the following steps:
  • the system for double-digestion of the pRNAT-U6.1 / Neo plasmid is as follows: 10 ⁇ FastDigeset Buffer, and the endo-digestion enzyme FastDigest Xma I and FastDigest BstBI, pRNAT-U6.1 / Neo plasmid.
  • the specific process steps include:
  • the step of transforming the ligation product into competent E. coli Stbl3 is as follows:
  • the plasmid expressing the LwCas13a gene provided by the present invention can replace the existing plasmid for RNA interference, and can induce RNA interference with higher specificity and lower off-target rate in cells, thereby providing better research for gene function. tool.
  • Figure 1 is a map of the plasmid pRNAT-LwCas13a-Neo expressing the LwCas13a gene
  • Figure 2 shows the results of TEX28 gene quantitative PCR detection of the experimental and control cells.
  • LwCas13a design the corresponding expression cassette so that it contains these three elements in sequence (LwCas13a gene-IRES sequence-NeoR / KanR gene, "-" represents sequential connection), and The 5 'and 3' ends are respectively provided with Xma I and BstBI digestion sites, and their sequences are shown in SEQ ID NO.2.
  • Shanghai Biotech was commissioned to synthesize the expression cassette by gene synthesis.
  • X-Digest Xma I and FastDigest BstBI respectively treated the pRNAT-U6.1 / Neo plasmid and the pUC57-LwCas13a plasmid containing the expression cassette obtained in Example 1, and the target fragment was recovered by agarose gel electrophoresis.
  • the target fragment recovered in the previous step was ligated with T4 ligase.
  • the ligation system (10 ⁇ l) was: pRNAT-U6.1 / Neo plasmid 50 ng digested, the expression cassette 217 ng, 10 ⁇ T4 DNA Ligase Buffer 1 ⁇ l, T4 DNA Ligase 1 ⁇ l, ddH2O made up to 10 ⁇ l; connection conditions: 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 plasmid pRNAT-LwCas13a-Neo expressing the LwCas13a gene, and its map is shown in Figure 1.
  • the crRNA targeting human TEX28 gene was designed according to the crRNA design rules of Cas13a, and BamHI and AflII restriction sites were added to its 5 'and 3' ends, respectively, and its sequence is shown in SEQ ID NO.3.
  • RNAT-LwCas13a-Neo plasmid and crRNA sequence Digestion of the pRNAT-LwCas13a-Neo plasmid and crRNA sequence with BamHI and AflII endonucleases respectively, agarose gel electrophoresis was used to recover the target fragment, and T4 DNA ligase was used to connect and transform the competent colon. Stbl3 and sequencing. The correct sequence is the new RNAi vector targeting the human TEX28 gene and named as pRNAT-LwCas13a-TEX28.
  • the correct strain was sequenced and identified in Example 3, and then 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 endotoxin-free pRNAT-LwCas13a-TEX28 vector.
  • A549 cells Cultivate A549 cells until the fusion rate of A549 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 In accordance with the instructions of the Lipofectamine 2000 kit, 4 ⁇ g of pRNAT-LwCas13a-TEX28 plasmid was introduced. 48 hours after transfection, 600 ⁇ g / mL was added. G418 screening for 10 days. After the screening was completed, the concentration of puromycin was reduced to 200 ⁇ g / ml and the cells were expanded.
  • A549 cells without any treatment were used as a control group, and the cells selected in Example 5 were used as an experimental group. After total RNA was extracted and reverse transcription was performed, the expression level of TEX28 gene was detected by fluorescent quantitative PCR. The results are shown in FIG. 2 . It can be seen that the expression level of the TEX28 gene in the cells of the experimental group is significantly lower than that in the control group, indicating that the new RNAi vector targeting the human TEX28 gene can achieve RNA interference to the TEX28 gene.
  • the plasmid expressing the LwCas13a gene provided by the present invention can replace the existing plasmid for RNA interference, and can induce RNA interference with higher specificity and lower off-target rate in cells, thereby providing better research for gene function. tool.

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Abstract

提供了一种表达LwCas13a基因的质粒pRNAT-LwCas13a-Neo，包括LwCas13a基因CDS序列与pRNAT-U6.1/Neo真核表达载体的重组构建而成，其核苷酸序列如SEQ ID NO.1所示。还提供了一种表达LwCas13a基因质粒的构建方法及其应用。所提供的表达LwCas13a基因的质粒可替代现有的用于RNA干扰的质粒，可在细胞中诱导产生特异性更高、脱靶率更低的RNA干扰。

Description

一种表达LwCas13a基因的质粒、构建方法及其应用 技术领域
本发明属于生物工程与生物技术领域，特别涉及一种表达LwCas13a基因的质粒、构建方法及其应用。
 背景技术
RNA干扰（RNAi）是指当细胞中导入与内源性mRNA编码区同源的双链RNA时，该mRNA发生降解而导致基因沉默的现象，是生物体内抑制特定基因表达的一种现象。外源导入或者由转基因、病毒感染等各种方式引入的双链RNA将被RNaseⅢ家族中的一个能够特异性识别双链RNA的酶Dicer，以ATP依赖的方式逐步切割为19～23nt的小分子干扰RNA片段(siRNA)。siRNA双链在RISC作用下双链解开，同源的靶目标RNA结合，在核酸内切酶的作用下，将靶目标mRNA切断，从而阻断基因表达。
 技术问题
现有的RNAi技术体系虽然已经成熟，且其效率较高，但其特异性较低和脱靶效应等问题却长期无法得到有效的解决，只能设计多对干扰序列并通过实验筛选出其中特异性和效率均较好的序列，步骤繁琐，耗时较长，现有技术并无较好的解决方案。
2016首次报道了一种RNA靶向的CRISPR酶——Cas13a。与CRISPR/Cas9切割DNA的活性不同，CRISPR/Cas13a能够用于切割细菌细胞中特定的RNA序列。研究表明，来自Leptotrichia wadei的LwCas13a能够以比现有RNAi工具更强的特异性在目标RNA上切割特定的位点。但在现有技术中，通常使用的是纯蛋白形式的LwCas13a，在crRNA的引导下对真核生物细胞内的RNA进行切割，尚未有通过真核表达载体表达LwCas13a进行RNA切割的报道。
 技术解决方案
本发明通过下列技术方案解决上述技术问题：
一种表达LwCas13a基因的质粒pRNAT-LwCas13a-Neo，包括LwCas13a基因CDS序列与 pRNAT-U6.1/Neo真核表达载体的重组构建而成，其核苷酸序列如SEQ ID NO.1所示。
一种构建所述表达LwCas13a基因质粒的方法，包括如下步骤：
（1）合成含有LwCas13a基因-IRES序列-NeoR/KanR基因的表达盒（“-”代表顺序连接），并在其5’和3’端分别设置Xma I和BstBI酶切位点，其序列如SEQ ID NO.2所示；
（2）用Xma I和BstBI酶对pRNAT-U6.1/Neo质粒进行双酶切，回收后获得线性化的pRNAT-U6.1/Neo质粒；
（3）将含有LwCas13a基因、IRES序列及NeoR/KanR基因的表达盒与线性化的pRNAT-U6.1/Neo质粒混匀后，用T4 DNA连接酶进行连接。连接产物转化感受态大肠杆菌Stbl3。
（4）大量培养经转化的Stbl3大肠杆菌，提取其中的重组质粒并委托测序。测序结果正确的即为所述的表达LwCas13a基因的质粒。
优选地，对pRNAT-U6.1/Neo质粒进行双酶切的体系如下：10×FastDigeset Buffer，内切酶FastDigest Xma I和FastDigest BstBI，pRNAT-U6.1/Neo质粒。具体的工艺步骤包括：
a. 配制反应体系：10×FastDigeset Buffer 2 μL +内切酶FastDigest Xma I和FastDigest BstBI各1 μL + pRNAT-U6.1/Neo质粒 1 μg，ddH2O补足至20 μL；
b. 将上述反应体系放置37℃水浴锅反应1 h；
c. 1%琼脂糖凝胶电泳观察结果并回收片段。
优选地，连接产物转化感受态大肠杆菌Stbl3步骤如下：
a. 取出储存于-80℃的感受态大肠杆菌Stbl3，冰浴解冻后加入10 μL连接产物，混匀，冰上静置30min；
b. 42℃热休克90 s，取出置冰上2 min；
c. 加入500 μL LB培养基，37℃、250rpm振荡培养45min；
d. 取出100 μL转化液涂于带有相应抗性的LB固体培养基上，37℃倒置培养8～12h。
 有益效果
本发明提供的表达LwCas13a基因的质粒可替代现有的用于RNA干扰的质粒，可在细胞中诱导产生特异性更高、脱靶率更低的RNA干扰，进而为基因功能的研究提供更好的工具。
 附图说明
图1为表达LwCas13a基因的质粒pRNAT-LwCas13a-Neo的图谱；
图2为实验组和对照组细胞的TEX28基因荧光定量PCR检测结果。
 本发明的实施方式
下面结合实施例，对本发明作进一步说明。下述实施例中，凡未注明具体实验条件的，均为按照本领域技术人员熟知的常规条件、实验室手册中的条件、按照制造厂商所建议的条件。
实施例 1 ：基因表达盒的设计
根据LwCas13a的序列、IRES序列及NeoR/KanR基因序列，设计相应的表达盒，使其顺序包含这三个元件（LwCas13a基因-IRES序列-NeoR/KanR基因，“-”代表顺序连接），并在其5’和3’端分别设置Xma I和BstBI酶切位点，其序列如SEQ ID NO.2所示。委托上海生工以基因合成的方式合成该表达盒。
实施例 2 ：表达 LwCas13a 基因的质粒 pRNAT-LwCas13a-Neo 的构建
用内切酶FastDigest Xma I和FastDigest BstBI分别处理pRNAT-U6.1/Neo质粒及实施例1获得的含有所述表达盒的pUC57-LwCas13a质粒，琼脂糖凝胶电泳回收目的片段。
将上一步回收的目的片段用T4连接酶进行连接，连接体系（10 μl）为：经酶切的pRNAT-U6.1/Neo质粒 50 ng，所述表达盒217 ng，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。
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表达LwCas13a基因的质粒pRNAT-LwCas13a-Neo，其图谱如图1所示。
实施例 3 ：靶向人 TEX28 基因的新型 RNAi 载体的制备
根据Cas13a的crRNA设计规则设计靶向人TEX28基因的crRNA，并在其5’端和3’端分别加上BamHI和AflII酶切位点，其序列如SEQ ID NO.3所示。
用BamHI和AflII内切酶分别对pRNAT-LwCas13a-Neo质粒和crRNA序列进行酶切，琼脂糖凝胶电泳回收目的片段并按照实施例2中的步骤用T4 DNA连接酶进行连接、转化感受态大肠杆菌Stbl3及测序。测序正确的即为所述靶向人TEX28基因的新型RNAi载体，命名为pRNAT-LwCas13a-TEX28。
实施例 4 ：无内毒素质粒的提取
取实施例3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pRNAT-LwCas13a-TEX28载体。
实施例 5 ： A549 细胞的转染
培养A549细胞，待A549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pRNAT-LwCas13a-TEX28质粒，转染后48 h，加入600 μg/mL G418筛选10 d。筛选完成后，将嘌呤霉素的浓度降为200 μg/ml继续扩大培养细胞。
实施例 6 ：荧光定量 PCR 检测 TEX28 基因的 RNAi 效果
以未经任何处理的A549细胞作为对照组，实施例5中筛选出的细胞为实验组，提取总RNA并进行逆转录后，荧光定量PCR检测TEX28基因的表达水平，其结果如图2所示。可以看到，实验组细胞的TEX28基因表达水平显著低于对照组细胞，说明所述靶向人TEX28基因的新型RNAi载体可以实现对TEX28基因的RNA干扰。
 工业实用性
本发明提供的表达LwCas13a基因的质粒可替代现有的用于RNA干扰的质粒，可在细胞中诱导产生特异性更高、脱靶率更低的RNA干扰，进而为基因功能的研究提供更好的工具。

Claims (4)

  1. 一种表达LwCas13a基因的质粒，其特征在于，包括LwCas13a基因CDS序列与 pRNAT-U6.1/Neo真核表达载体的重组构建而成，其核苷酸序列如SEQ ID NO.1所示。
  2. 一种构建如权利要求1所述的表达LwCas13a基因质粒的方法，其特征在于，包括如下步骤：
    （1）合成含有LwCas13a基因-IRES序列-NeoR/KanR基因的表达盒（“-”代表顺序连接），并在其5’和3’端分别设置Xma I和BstBI酶切位点；
    （2）用Xma I和BstBI酶对pRNAT-U6.1/Neo质粒进行双酶切，回收后获得线性化的pRNAT-U6.1/Neo质粒进行；
    （3）将含有LwCas13a基因、IRES序列及NeoR/KanR基因的表达盒与线性化的pRNAT-U6.1/Neo质粒混匀后，用T4 DNA连接酶进行连接。连接产物转化感受态大肠杆菌Stbl3。
    （4）大量培养经转化的Stbl3大肠杆菌，提取其中的重组质粒并委托测序。测序结果正确的即为所述的表达LwCas13a基因的质粒。
  3. 根据权利要求2所述的一种构建如权利要求1所述的表达LwCas13a基因质粒的方法，其特征在于：步骤（3）中所述连接产物转化感受态大肠杆菌Stbl3步骤如下：
    a. 取出储存于-80℃的感受态大肠杆菌Stbl3，冰浴解冻后加入10 μL连接产物，混匀，冰上静置30min；
    b. 42℃热休克90 s，取出置冰上2 min；
    c. 加入500 μL LB培养基，37℃、250rpm振荡培养45min；
    d. 取出100μl转化液涂于带有相应抗性的LB固体培养基上，37℃倒置培养8～12h。
  4. 根据权利要求2所述的一种构建如权利要求1所述的表达LwCas13a基因质粒的方法，其特征在于：步骤（2）中所述的双酶切体系如下：10×FastDigeset Buffer，内切酶FastDigest Xma I和FastDigest BstBI，pRNAT-U6.1/Neo质粒。具体的工艺步骤包括：
    a. 配制反应体系：10×FastDigeset Buffer 2 μL +内切酶FastDigest Xma I和FastDigest BstBI各1 μL + pRNAT-U6.1/Neo质粒 1 μg，ddH2O补足至20 μL；
    b. 将上述反应体系放置37℃水浴锅反应1 h；
    c. 1%琼脂糖凝胶电泳观察结果并回收片段。
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WO2018005873A1 (en) * 2016-06-29 2018-01-04 The Broad Institute Inc. Crispr-cas systems having destabilization domain
CN107557455A (zh) * 2017-09-15 2018-01-09 国家纳米科学中心 一种基于CRISPR‑Cas13a的特异性核酸片段的检测方法
CN108048466A (zh) * 2017-12-21 2018-05-18 嘉兴市第医院 CRISPR-Cas13a系统特异性靶向人RSPO2基因的crRNA及系统和应用

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* Cited by examiner, † Cited by third party
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WO2017219027A1 (en) * 2016-06-17 2017-12-21 The Broad Institute Inc. Type vi crispr orthologs and systems
WO2018005873A1 (en) * 2016-06-29 2018-01-04 The Broad Institute Inc. Crispr-cas systems having destabilization domain
CN107557455A (zh) * 2017-09-15 2018-01-09 国家纳米科学中心 一种基于CRISPR‑Cas13a的特异性核酸片段的检测方法
CN108048466A (zh) * 2017-12-21 2018-05-18 嘉兴市第医院 CRISPR-Cas13a系统特异性靶向人RSPO2基因的crRNA及系统和应用

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