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WO2019237380A1 - 一种基于慢病毒的 CRISPR/Cpf1 基因编辑载体及其应用 - Google Patents

一种基于慢病毒的 CRISPR/Cpf1 基因编辑载体及其应用 Download PDF

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WO2019237380A1
WO2019237380A1 PCT/CN2018/091710 CN2018091710W WO2019237380A1 WO 2019237380 A1 WO2019237380 A1 WO 2019237380A1 CN 2018091710 W CN2018091710 W CN 2018091710W WO 2019237380 A1 WO2019237380 A1 WO 2019237380A1
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lentivirus
vector
cpf1
gene editing
gene
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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 WO2019237380A1 publication Critical patent/WO2019237380A1/zh

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    • 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
    • C12N15/86—Viral vectors
    • C12N15/867—Retroviral vectors

Definitions

  • the invention relates to the fields of gene editing and molecular biology, and specifically discloses a lentivirus-based CRISPR / Cpf1 gene editing vector and application thereof.
  • CRISPR / Cas9 technology Since Jinek et al. Demonstrated in 2012 that the CRISPR / Cas9 system can bind to specific sites of DNA target sequences and cut in vitro, CRISPR / Cas9 technology has brought a huge impact to the biological field. In 2013, it was published by Science Magazine. Named one of the top ten scientific breakthroughs, and topped the list in 2015. Using CRISPR / Cas9 technology, scientists can efficiently and accurately trim, replace or add DNA sequences, and can quickly achieve microbial genome editing, animal and plant species optimization, animal model construction, and even promote a disruptive revolution in disease treatment. .
  • the CRISPR / Cas9 system has also exposed its defects and limitations, such as severe off-target effects, and can only identify GC-rich PAM sites.
  • Cpf1 V-type CRISPR effector protein
  • Cpf1 Compared with Cas9, Cpf1 has lower non-specific and off-target effects; Cpf1 only needs a segment of single-stranded RNA consisting of 42-44 nucleotides to recognize and cut DNA, which simplifies the experimental design steps and is more conducive to Multi-gene editing; Cpf1 can recognize AT-rich PAM sites, which can expand the editing range of CRISPR; In addition, Cpf1 cleavage will produce sticky ends, which can promote the insertion of target genes into target loci through non-homologous recombination.
  • the development of CRISPR / Cpf1 is helpful to break through and overcome some limitations in CRISPR / Cas9 applications, so it is called a new generation of CRISPR genome editing tools.
  • the invention provides a lentivirus-based CRISPR / Cpf1 gene editing vector and its application to solve the above problems.
  • a lentivirus-based CRISPR / Cpf1 gene editing vector the full sequence of which is as SEQ ID No.1.
  • the above vector is based on the pLVX-shRNA1 plasmid, and the entire expression frame composed of the PGK promoter-puromycin resistance gene is replaced with the expression frame composed of the CAG promoter-Cpf1 gene-nuclear localization sequence-2A peptide-puromycin resistance gene. Its sequence is shown in SEQ ID No.2.
  • the Cpf1 gene is AsCpf1 and its sequence is shown in SEQ ID No. 3; the 2A peptide is T2A and its sequence is shown in SEQ ID No. 4.
  • the synthetic sequence and pLVX-shRNA1 vector were treated with EcoRI and MluI, respectively, and the digested product was recovered by electrophoresis to obtain the expression frame and linearized pLVX-shRNA1 vector.
  • the ligation product is transformed into E. coli.
  • a large number of transformed E. coli were cultured and the recombinant plasmids were extracted.
  • the correct sequence was identified as the lentivirus-based CRISPR / Cpf1 gene editing vector pLVX-AsCpf1-puro, and its map is shown in FIG. 1.
  • the lentivirus-based CRISPR / Cpf1 gene editing vector provided by the present invention can conveniently and efficiently perform gene editing on various cell lines (especially difficult-to-transfect cell lines) and accelerate related research processes.
  • Figure 1 is a vector map of pLVX-AsCpf1-puro
  • the pLVX-shRNA1 lentiviral vector used in the examples of the present invention was purchased from Clontech, and the Stbl3 competent E. coli used was purchased from Beijing Quanshijin, and the reagents used were all commercially available products.
  • the synthetic sequence is integrated into the pUC57 vector.
  • This vector and pLVX-shRNA1 vector were digested with EcoRI and MluI endonucleases respectively, and the desired fragments were recovered after agarose gel electrophoresis to obtain a purified expression frame sequence and a linearized pLVX-shRNA1 vector.
  • the ligation product is transformed into E. coli Stbl3.
  • a large number of transformed E. coli were cultured and the recombinant plasmids were extracted.
  • the correct sequence was identified as the lentivirus-based CRISPR / Cpf1 gene editing vector pLVX-AsCpf1-puro.
  • Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System provides crRNA design rules and gRNA sequences that target the DNMT1 gene, design crRNAs that target the DNMT1 gene, and according to the actual situation of the pLVX-AsCpf1-puro vector in its 5 ' The sticky ends of BamHI and EcoRI digestion sites were added at the 3 'and 3' ends, respectively.
  • the forward sequence was 5'-GATCCTAATTTCTACTCTTGTAGATCTGATGGTCCATGTCTGTTACTCG-3 ', and the reverse sequence was 5'- GGAGTAACAGACATGGACCATCAGATCTACAAGAGTAGAAATTAAATTC-3'.
  • 5 ⁇ L of each was mixed, heated at 95 ° C. for 5 min, and then naturally cooled to room temperature to form a double-stranded DNA with sticky ends of BamHI and EcoRI.
  • T4 DNA ligase was used to ligate a and b products.
  • the ligated product was then transformed into E. coli Stbl3 and identified by sequencing.
  • the recombinant vector contained in the correct strain was the pLVX-AsCpf1-puro-DNMT1 vector.
  • Example 2 Take the sequence of Example 2 to identify the correct strain, and place it in LB liquid medium with an Ampicillin concentration of 100 ⁇ g / ml, and culture it at 250 rpm and 37 ° C with shaking for 12-16 h. 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 pLVX-AsCpf1-puro-DNMT1 vector.
  • 293T cells were cultured and transfected after two passages of growth and culture: pLVX-AsCpf1-puro-DNMT1 vector was taken and transfected with the packaging plasmid and transfection reagent provided by the integrase-deficient Lenti-X HTX lentiviral packaging system Stained in 293T cells. 48 hours before transfection, inoculate cells into a well plate or petri dish for lentivirus production. During transfection, the confluence of cells is about 70% -80% is the best infection state, and the viability is ⁇ 95%. The staining time was the starting point. The supernatants were harvested after 48 h and 72 h, filtered through a 0.45 ⁇ m filter and stored at -80 ° C.
  • Untreated 293T cells control group
  • lentivirus-treated 293T cells experimental group
  • genomic DNA was extracted, and then the high-fidelity PCR enzyme PrimeSTAR HS was used to expand The gene editing site is expected to be amplified, and the PCR product is recovered by electrophoresis.
  • the lentivirus-based CRISPR / Cpf1 gene editing vector provided by the present invention can conveniently and efficiently perform gene editing on various cell lines (especially difficult-to-transfect cell lines) and accelerate related research processes.

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Abstract

一种基于慢病毒的CRISPR/Cpf1基因编辑载体及其应用。在常规慢病毒载体pLVX-shRNA1基础上，将PGK启动子-puromycin抗性基因组成的表达框整体替换为CAG启动子-Cpf1基因-2A肽-puromycin抗性基因组成的表达框，获得一种可用于Cpf1基因编辑的重组慢病毒载体。通过慢病毒包装程序获得的重组慢病毒，能够体外感染目的细胞并对目的基因进行编辑。

Description

一种基于慢病毒的CRISPR/Cpf1基因编辑载体及其应用 技术领域
本发明涉及基因编辑和分子生物学领域，具体地公开了一种基于慢病毒的CRISPR/Cpf1基因编辑载体及其应用。
 背景技术
近年来，基因编揖技术领域发展迅速，新型基因编揖工具不断被开发，尤其是CRISPR基因编辑系统的出现，使基因编辑技术进入了新的时代｡目前基因工程定点基因编辑工具中，可设计目标序列的定点核酸内切酶主要有三种，即：ZFN（锌指核酸酶），TALEN（转录激活子样效应因子核酸酶）和CRISPR（规律成簇的间隔短回文重复）系统｡其中CRISPR系统，因其设计构建方便，敲除效率高，应用范围广等优势，被广泛应用于基因编辑研究中｡
自Jinek等在2012年证实了CRISPR/Cas9系统可以在体外条件下与DNA靶序列的特定位点结合并进行剪切，CRISPR/Cas9技术为生物领域带来了巨大的冲击，2013年被Science杂志评为十大科学突破之一，2015年更是荣登榜首。利用CRISPR/Cas9技术，科学家们能够高效、精确地对DNA序列迚行修剪、替换或添加，可以快速地实现微生物基因组编辑、动植物的品种优化、动物模型构建，甚至推动疾病治疗的颠覆性革命。
 技术问题
然而，随着研究的不断深入，CRISPR/Cas9系统也暴露了它的缺陷和局限性，例如严重的脱靶效应，只能识别富含GC的PAM位点等。
2015年美国MIT的FengZhang小组报道了一种新的2类V型CRISPR效应蛋白Cpf1，是在单链向导RNA引导下与靶DNA特定位点结合并切割的核酸内切酶。与Cas9相比，Cpf1具有更低的非特异性和脱靶效应；Cpf1只需一段42-44个核苷酸组成的单链RNA即可识别和剪切DNA，从而简化了实验设计步骤，更有利于多基因编辑；Cpf1能够识别富含AT的PAM位点，可以扩展CRISPR的编辑范围；此外，Cpf1剪切会产生黏性末端，可促进目的基因通过非同源重组的方式插入靶定位点。CRISPR/Cpf1的开发有利于突破和克服CRISPR/Cas9应用中的一些限制，因此被称为是新一代的CRISPR基因组编辑工具。但现有技术中缺乏基于慢病毒的CRISPR/Cpf1基因编辑载体，阻碍了CRISPR/Cpf1系统在难转染细胞中的应用，拖慢了相关研究的进程。
 技术解决方案
本发明提供一种基于慢病毒的CRISPR/Cpf1基因编辑载体及其应用以解决上述问题。
一种基于慢病毒的CRISPR/Cpf1基因编辑载体，其全序列如SEQ ID No.1所示。
上述载体是在pLVX-shRNA1质粒的基础上，将PGK启动子-puromycin抗性基因组成的表达框整体替换为CAG启动子-Cpf1基因-核定位序列-2A肽-puromycin抗性基因组成的表达框，其序列如SEQ ID No.2所示。
上述载体中，所述Cpf1基因为AsCpf1，其序列如SEQ ID No.3所示；所述2A肽为T2A，其序列如SEQ ID No.4所示。
上述载体的构建方法如下：
1、设计CAG启动子-AsCpf1基因-T2A -puromycin抗性基因组成的表达框，并分别在其5’端和3’端加上EcoRI和MluI酶切位点，其序列如SEQ ID No.2所示。委托合成该序列。
2、用EcoRI和MluI分别处理合成序列及pLVX-shRNA1载体，电泳回收酶切产物，得到表达框及线性化的pLVX-shRNA1载体。
3、将线性化的pLVX-shRNA1载体与表达框序列按物质的量之比为1：5的比例混合（其中线性化的pLVX-shRNA1载体为50 μg），然后用T4 DNA连接酶连接过夜。
4、连接产物转化大肠杆菌。大量培养转化后的大肠杆菌并提取其中的重组质粒，经测序鉴定正确的即为所述基于慢病毒的CRISPR/Cpf1基因编辑载体pLVX-AsCpf1-puro，其图谱如图1所示。
 有益效果
本发明提供的基于慢病毒的CRISPR/Cpf1基因编辑载体可方便、高效地对各种细胞系（尤其是难转染细胞系）进行基因编辑，加速相关研究进程。
 附图说明
图1为pLVX-AsCpf1-puro的载体图谱；
图2对照组和实验组293T细胞的T7 Endonuclease I试验结果图，其中：M-Marker，1-实验组，2-对照组。
 本发明的实施方式
以下所述是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也视为本发明的保护范围。
下述实施例中所用的方法如无特别说明均为常规方法。所用引物及DNA 序列均由上海 生工公司合成。
本发明实施例所使用的pLVX-shRNA1慢病毒载体购自Clontech，所使用的Stbl3感受态大肠杆菌购自北京全式金，所使用的试剂均为市售商品。
实施例 1 ： pLVX-AsCpf1-puro 载体的构建
a. 设计CAG启动子-AsCpf1基因-T2A -puromycin抗性基因组成的表达框，并分别在其5’端和3’端加上EcoRI和MluI酶切位点，其序列如SEQ ID No.2所示。委托合成该序列。
b. 合成的序列整合在pUC57载体中。用EcoRI和MluI内切酶分别对该载体和pLVX-shRNA1载体进行酶切，琼脂糖凝胶电泳后回收所需目的片段，得到纯化的表达框序列和线性化的pLVX-shRNA1载体。
c. 将线性化的pLVX-shRNA1载体和纯化的表达框序列按物质的量之比为1:5为混合（其中线性化的pLVX-shRNA1载体为50 μg），然后用T4 DNA连接酶连接过夜。
d. 连接产物转化大肠杆菌Stbl3。大量培养转化后的大肠杆菌并提取其中的重组质粒，经测序鉴定正确的即为所述基于慢病毒的CRISPR/Cpf1基因编辑载体pLVX-AsCpf1-puro。
实施例 2 ：靶向人 DNMT1 基因的 pLVX-AsCpf1-puro-DNMT1 载体的制备
a. 根据Bernd Zetsche等在其文章（Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System）中提供crRNA设计规则及靶向DNMT1基因的gRNA序列，设计靶向DNMT1基因的crRNA，并根据pLVX-AsCpf1-puro载体的实际情况在其5’端和3’端分别添加BamHI和EcoRI酶切位点的粘性末端，其正向序列为5’-GATCCTAATTTCTACTCTTGTAGATCTGATGGTCCATGTCTGTTACTCG-3’，其反向序列为：5’- GGAGTAACAGACATGGACCATCAGATCTACAAGAGTAGAAATTAAATTC-3’，合成这两段序列。溶解后，各取5μL混匀后，95℃加热5 min，然后自然冷却至室温，形成带有BamHI和EcoRI粘性末端的DNA双链。
b. 用BamHI和EcoRI酶切pLVX-AsCpf1-puro载体，PCR Cleanup试剂盒回收线性化的pLVX-AsCpf1-puro载体。
c. 用T4 DNA连接酶连接a、b两步获得的产物。然后将连接产物转化至大肠杆菌Stbl3中，测序鉴定。鉴定正确的菌株所含的重组载体即为pLVX-AsCpf1-puro-DNMT1载体。
实施例 3 ：无内毒素质粒 DNA 的制备
取实施例2测序鉴定正确的菌株，置Ampicillin浓度为100μg/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pLVX-AsCpf1-puro-DNMT1载体。
实施例 4 ：慢病毒的包装
培养293T细胞，待生长培养传代2次后，进行转染操作：取pLVX-AsCpf1-puro-DNMT1载体，用整合酶缺陷型的Lenti-X HTX慢病毒包装系统提供的包装质粒和转染试剂转染293T细胞中。转染前48小时，接种细胞至备用生产慢病毒的孔板或是培养皿中，转染时，细胞汇合度约为70%-80%为最佳感染状态，活力≥95%以上；以转染时间为起始点，分别于48 h和72 h后收获上清，0.45 μm滤膜过滤后，保存于-80℃下。
实施例 5 ： 293T 细胞的慢病毒感染及嘌呤霉素筛选
培养293T细胞至其汇合度约为70%-80%时，加入慢病毒与培养基的混合液（含4 μg/mL polybrene）处理24 h后，将慢病毒液换成含1 μg/mL嘌呤霉素的完全培养基，开始进行筛选培养，筛选时间为7 d。隔天换液一次。被慢病毒感染的细胞将形成单细胞克隆，此时即完成了细胞的筛选。
实施例 6 ： T7 Endonuclease I 试验检测转导效果
分别取未经处理的293T细胞（对照组）和经慢病毒处理后的293T细胞（实验组）接种至六孔板，待细胞长满后，提取基因组DNA，然后应用高保真PCR酶PrimeSTAR HS扩增基因编辑预期发生的位点，电泳回收PCR产物。
PCR产物经重退火后，用T7 Endonuclease I在37℃酶切1 h，然后进行琼脂糖凝胶电泳，结果如图2所示，与对照组相比，实验组出现了2条明显的切割条带，表明pLVX-AsCpf1-puro-DNMT1载体包装成的慢病毒可对人DNMT1基因进行编辑，说明pLVX-AsCpf1-puro载体可用于细胞的基因编辑研究中。
 工业实用性
本发明提供的基于慢病毒的CRISPR/Cpf1基因编辑载体可方便、高效地对各种细胞系（尤其是难转染细胞系）进行基因编辑，加速相关研究进程。

Claims (3)

  1. 一种基于慢病毒的CRISPR/Cpf1基因编辑载体，其特征在于，该载体的全序列如SEQ ID No.1所示。
  2. 根据权利要求1所述的一种基于慢病毒的CRISPR/Cpf1基因编辑载体，其特征在于，在pLVX-shRNA1质粒的基础上，将PGK启动子-puromycin抗性基因组成的表达框整体替换为CAG启动子-Cpf1基因-2A肽-puromycin抗性基因组成的表达框，其序列如SEQ ID No.2所示。
  3. 根据权利要求2所述的一种基于慢病毒的CRISPR/Cpf1基因编辑载体，其特征在于，所述Cpf1基因为AsCpf1，其序列如SEQ ID No.3所示；所述2A肽为T2A，其序列如SEQ ID No.4所示。
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