CN107604003A - 一种基于线性化crispr‑cas9慢病毒载体基因敲除试剂盒及其应用 - Google Patents
一种基于线性化crispr‑cas9慢病毒载体基因敲除试剂盒及其应用 Download PDFInfo
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Abstract
本发明公开了一种基于CRISPR‑CAS9慢病毒载体的基因敲除试剂盒，该试剂盒包括线性化的CRISPR‑CAS9慢病毒载体和对应的T4连接酶及感受态细胞，其特征在于所述的线性化的CRISPR‑CAS9慢病毒载体由核酸内切酶处理的环状载体构成，所述的T4连接酶是优化的T4连接酶，所述的感受态细胞是具有抑制重组特性的Stbl3感受态。该试剂盒能直接进行CRISPR‑CAS9慢病毒载体的构建，其比国内外商品化试剂盒操作方便、且构建重组得速度快，可有效提高目前基于CRISPR‑CAS9慢病毒载体的构建效率，降低基因敲除的费用。
Description
技术领域
本发明涉及生物领域，具体涉及一种研究用配制品，特别是用于基于CRISPR-CAS9慢病毒载体的基因敲除试剂盒。
背景技术
基因修饰是目前生物领域的重要问题，近年来，基因组编辑技术的快速发展为生物学研究带来了新纪元。与传统的基因克隆技术不同，基因组编辑技术可直接在基因组上进行DNA序列的敲除、插人、定点突变以及组合编辑等，实现基因功能与调控元件的系统研究，在工业生物工程等方面具有广阔的应用前景。早期，基因组编辑技术主要利用同源重组介导的打靶技术，但由于效率较低，极大地限制了其应用。为解决这一难题，一系列人工核酸内切酶介导的基因组编辑技术被开发，可通过在基因组特定位置上形成DNA双链断裂，借助于细胞自身的修复系统如非同源末端连接或同源重组，从而实现在不同生物与细胞类型中有效的定点基因组编辑。目前，主要有4种不同的人工核酸内切酶已应用于基因组编辑：巨核酶技术、锌指核酸内切酶(ZFN)、类转录激活因子效应物核酸酶(TALEN)。与RNA靶向DNA内切酶Cas9、ZFN与TALEN均可通过蛋白一DNA相互作用识别基因组上的特定DNA序列。巨核酶技术具有核酸内切酶结构域与DNA结合域，而ZFN与TALEN分别具有可识别3个与1个核苷酸的DNA结合域，需与核酸内切酶FokI的内切酶结构域形成融合蛋白后，完成基因组特定位点的切割。但这些技术各有不足，如巨核酶技术的氨基酸残基与DNA靶序列之间无明确特异性；ZFN的DNA结合域则会受DNA靶序列上下游的影响，需通过构建不同的融合蛋白来定位到不同的DNA序列，构建操作较为繁琐，成本较高，脱靶风险较高。RNA靶向内切酶CRISPR—Cas9介导的基因组编辑技术则通过一段短的引导RNA(guideRNA)来识别特定的DNA序列，只需通过改变这段引导RNA序列即可使Cas9定位到新的DNA序列。该技术已被应用于多种生物，包括人、小鼠、大鼠、斑马鱼、秀丽隐杆线虫、植物及细菌。其技术也不断被拓展，如利用多个引导RNA序列可同时进行基因组上多个不同位点的编辑；将其DNA结合域与不同转录调控蛋白相融合，可实现对特定基因的转录激活或抑制；将对特定序列的调控蛋白模块与基因组或表观基因组的修饰酶相融合，则可实现对基因组的动态控制。
慢病毒是目前转染领域运用最多的一种技术，通过构建慢病毒包装质粒可以高效的将目的基因整合到细胞的染色体上，实现难转染细胞的高效转染和达到降低昂贵转染试剂的效果。
CRISPR—Cas9介导的基因组编辑技术以其操作简单、特异性高的特点成为研究热点,而目前仍缺少商业化的试剂盒用于基因编辑技术的操作，因此研发准确可靠、简单实用、适合各种研究者操作的CRISPR-CAS9慢病毒载体基因敲除试剂盒为当前迫切之需，本发明结合慢病毒载体的高效转染特性和CRISPR-CAS9技术的高效基因编辑特征，将解决该领域的重要难题。
发明内容
本发明要解决的技术问题是控制慢病毒载体的基因敲除的效率，缩小基因敲除的差异性。
本发明解决上述问题的技术方案是：
一种基于CRISPR-CAS9慢病毒载体基因敲除试剂盒，该试剂盒包括线性化CRISPR-CAS9慢病毒载体、T4连接酶和T4连接酶的缓冲液、高转化性Stbl3感受态细胞，其特征在于：
1.所述的线性化CRISPR-CAS9慢病毒载体由CRISPR-CAS9慢病毒载体经核酸内切酶得到；
2.所述的连接酶是T4连接酶；
3.所述的感受态细胞为氯化钙法制备的高转化性Stbl3感受态细胞。
本发明试剂盒中，线性化的CRISPR-CAS9慢病毒载体包括但不仅限于慢病毒载体，也可以用于普通CRISPR-CAS9载体；T4连接酶为常用的或商业化的T4连接酶；Stbl3感受态细胞是通过氯化钙法制备而成。
本发明试剂盒制备基于CRISPR-CAS9慢病毒载体的敲除质粒是通过分子克隆的方式实现的，即通过构建目的基因的引导RNA序列，重组进入线性化CRISPR-CAS9慢病毒载体而成，具体过程如下：
1.CRISPR-CAS9慢病毒载体的线性化过程中，总体积最好为50μl，其中CRISPR-CAS9慢病毒载体为2μg，BsmBI酶2μl，BsmBI酶缓冲液5μl，剩余用灭菌去离子水补足至50μl。然后在37℃放置1小时，然后进行切胶回收，具体的酶切步骤如下：
2μg CRISPR-CAS9慢病毒环状载体
2μl BsmBI酶
5μl BsmBI酶缓冲液(10U/μl)
41μl灭菌去离子水
50μl总体积
酶切后载体在1％的琼脂糖凝胶进行电泳后，核酸染料染色后在紫外灯下进行观察，切取不超过400mg的1％的琼脂糖凝胶，55℃溶解后进行载体回收，回收后的载体进行浓度测定。
发明人通过调节载体量和BsmBI酶的用量，以酶切效率进行评价，筛选出了酶切效率最高的配比。
线性化CRISPR-CAS9慢病毒载体的制备过程中条件的筛选
| 载体量(μg) | BsmBI酶(μl) | 总体积(μl) | 酶切效率(％) |
| 1 | 1 | 20 | 55 |
| 1 | 2 | 20 | 70 |
| 2 | 1 | 20 | 50 |
| 2 | 2 | 20 | 65 |
| 1 | 5 | 50 | 95 |
| 1 | 10 | 50 | 90 |
| 2 | 5 | 50 | 98 |
| 2 | 10 | 50 | 80 |
2.构建目的基因的引导RNA序列，gRNA靶点选择及其寡核苷酸链合(http://crispr-mit.edu/网站)sgRNA靶点选择及其寡核苷酸链合成：应用设计标准如下：正义链模板的5’端添加CACC；反义链模板的5’端添加AAAC。
将sgRNA寡核苷酸单链退火形成双链：取等量的正义链和反义链混合(终浓度为10μl)。程序如下：37℃，30分钟；95℃，5分钟；94℃，12sec，-l℃/循环，70个循环；最终为24℃，具体反应的样品体积和浓度如下。
1μl正义链模板(100mM)
1μl反义链模板(100mM)
1μl T4连接酶缓冲液
7.5μl灭菌去离子水
10μl总体积
3.目的基因和线性化CRISPR-CAS9慢病毒载体的重组，将上述得到的正义链和反义链稀释至0.5μmoL/L。加入(1)中得到的线性化CRISPR-CAS9载体10ng，加入T4连接酶连接20分钟。转化高转化性Stbl3感受态，挑取单克隆，具体流程为：
1μl线性化CRISPR-CAS9载体10ng
1μl正义链和反义链混合物
1μl T4连接酶缓冲液
1μl T4连接酶(20U/μl)
加入灭菌去离子水至总体积10μl。
发明人改变T4连接酶体积，并通过连接效率来确定T4连接酶用量。连接效率的检测方法为进行连接反应后转化细菌，跟同样含量的对照质粒比较长出来多少细菌斑点。
T4连接酶和缓冲液组合的筛选
| T4连接酶体积(μl) | T4连接酶占比(％) | 连接效率(％) |
| 0.5 | 5 | 80 |
| 1 | 10 | 95 |
| 1.5 | 15 | 95 |
| 2 | 20 | 95 |
4.慢病毒载体测序
将挑取得到的单克隆在Stbl3细菌培养基中进行扩大培养，同时加入氨苄青霉素进行抗性筛选，将收集得到的Stbl3细菌进行裂解，抽提其中的重组质粒进行测序。测序成功的载体显示重组克隆构建成功。
Stbl3感受态与DH5感受态细胞对比实验
本发明试剂盒基于线性化CRISPR-CAS9慢病毒载体构建基因敲除的试剂盒具有以下优点：
(1)线性化CRISPR-CAS9慢病毒载体消除了不同实验者对载体的不一致现象，减少了不同反应的干扰，节省了载体处理的时间，提高了实验效率；
(2)本发明中提供的T4连接酶和缓冲液是经过多种测试得到的最优的一种组合，避免了不同品牌之间连接效率的不同，提高了实验的一致性；
(3)用本发明氯化钙法构建的高感染性Stbl3感受态，相比于传统的DH5感受态细胞减少了重组的发生。
4.采用本发明，使用者只需要合成目的基因的正、反链引物就可以立即进行基因敲除载体的构建，具有省时，省力，减低成本的效果。
附图说明
图1是线性化CRISPR-CAS9慢病毒载体的琼脂糖凝胶电泳图，其中显示的是线性化的载体，及经过BsmBI核酸酶酶切后的片段。由于载体里面有一段2000bp的插入序列，酶切后该序列被切割下来，通过该被切割下来的2000bp的序列可以证明得到线性化的CRISPR-CAS9慢病毒载体，方便回收。
图2是成功转化Stbl3后的重组质粒涂板后情况。
图3为转化到细胞中的情况，带有绿色荧光的显示出转染成功，方便后续实验。
具体实施方式
下述实施例1所述的普通克隆重组法为采用常用的基因所建立的CRISPR-CAS9慢病毒载体构建的基因敲除，该载体上带有绿色荧光标记的CRISPR-CAS9载体构建的基因敲除方法。
实施例1(普通克隆重组法构建的具有嘌呤霉素抗性的CRISPR-CAS9慢病毒载体基因敲除，以SNX10基因为例)
1、小鼠SNX10基因sgRNA设计：
(1)设计获得SNX10基因的正、反义链引物序列：
sgRNA1F：AAACGCAACGCATTACTTTGGAGTC SEQ ID No.1
sgRNA1R：CACCGCACGTGGATCAGCGTCGCCA SEQ ID No.2
sgRNA2F：CACCGCACGTGGATCAGCGTCGCCA SEQ ID No.3
sgRNA2R：AAACTGGCGACGCTGATCCACGTGC SEQ ID No.4
(2)试剂盒所述的线性化具有嘌呤霉素抗性的CRISPR-CAS9慢病毒载体；
(3)T4连接酶和T4连接酶缓冲液；
(4)高转化活性Stbl3感受态；
2、具有嘌呤霉素抗性的CRISPR-CAS9慢病毒克隆载体的重组
(1)将正义链和反义链退火形成双链：取等量的正义链和反义链混合(终浓度为10μM)。程序如下：37℃30分钟；95℃，5分钟；94℃，12sec，-l℃/循环，70个循环；最终为24℃，具体反应的样品体积和浓度如下。
1μl正义链模板(100mM)
1μl反义链模板(100mM)
1μl T4连接酶缓冲液
加入灭菌去离子水至总体积10μl。
反应完成后，在10μl中加入2ml灭菌去离子水。
(2)将上述得到的正义链和反义链混合物和试剂盒中的线性化CRISPR-CAS9载体加入试剂盒中的T4连接酶连接，连接20分钟。具体方法是：
1μl线性化的具有嘌呤霉素抗性的CRISPR-CAS9载体
1μl正义链和反义链混合物
1μl T4连接酶缓冲液
1μl T4连接酶
加入灭菌去离子水至总体积10μl。
(3)将本试剂盒中高转化活性的Stbl3感受态在冰上解冻，将上述11μl的正义链和反义链混合物加入感受态细胞并摇匀，冰上放置约30分钟。水浴42℃热激90秒后，加入1毫升LB培养基(氯化钠10g，蛋白胨10g；酵母提取物5g，加水1升灭菌后使用)，于37℃下170转/分钟摇床中培养1小时后，5000转/分钟离心1分钟。将沉淀涂在含有抗生素活性的LB琼脂糖(氯化钠10g，蛋白胨10g；酵母提取物5g，琼脂粉3g，加水1升灭菌后使用)板上，待生长16小时后取出，挑单克隆菌落。
3、CRISPR-CAS9慢病毒克隆载体测序，将上述挑选的单克隆菌落加入LB培养基中进行进行扩大培养，同时加入氨苄青霉素抗生素(100g/ml)进行抗性筛选，37℃下220转/分钟摇床中培养12小时后，抽提质粒测序。
测序结果显示重组克隆质粒中，以上两个正义链模板已经插入到载体中：
重组克隆构建成功。
实施例2(普通克隆重组法构建的具有嘌呤霉素抗性的CRISPR-CAS9慢病毒载体基因敲除，以HSF1基因为例)
1、人HSF1基因sgRNA设计：
(1)设计获得HSF1基因的正、反义链引物序列：
sgRNA1F：TCGTGAGCGACCCGGACACCGTTTT SEQ ID No.5
sgRNA1R：GGTGTCCGGGTCGCTCACGACGGTG SEQ ID No.6
sgRNA2F：CAGCTTCCACGTGTTCGACCGTTTT SEQ ID No.7
sgRNA2R：GGTCGAACACGTGGAAGCTGCGGTG SEQ ID No.8
sgRNA3F：GGAGTCAATGAGGGCGGTCGGTTTT SEQ ID No.9
sgRNA3R：CGACCGCCCTCATTGACTCCCGGTG SEQ ID No.10
(2)试剂盒所述的线性化具有嘌呤霉素抗性的CRISPR-CAS9慢病毒载体；
(3)T4连接酶和T4连接酶缓冲液；
(4)高转化活性Stbl3感受态；
2、具有嘌呤霉素抗性的CRISPR-CAS9慢病毒克隆载体的重组
(1)将正义链和反义链退火形成双链：取等量的正义链和反义链混合(终浓度为10μM)。程序如下：37℃，30分钟；95℃，5分钟；94℃，12sec，-l℃/循环，70个循环；最终为24℃，具体反应的样品体积和浓度如下。
1μl正义链模板(100mM)
1μl反义链模板(100mM)
1μl T4连接酶缓冲液
加入灭菌去离子水至总体积10μl。
反应完成后，在10μl中加入2ml灭菌去离子水。
(2)将上述得到的正义链和反义链混合物和试剂盒中的线性化CRISPR-CAS9载体加入试剂盒中的T4连接酶连接，连接20分钟。具体方法是：
1μl线性化的具有嘌呤霉素抗性的CRISPR-CAS9载体
1μl正义链和反义链混合物
1μl T4连接酶缓冲液
1μl T4连接酶
加入灭菌去离子水至总体积10μl。
(3)将本试剂盒中高转化活性的Stbl3感受态在冰上解冻，将上述11μl的正义链和反义链混合物加入感受态细胞并摇匀，冰上放置约30分钟。水浴42℃热激90秒后，加入1毫升LB培养基(氯化钠10g，蛋白胨10g；酵母提取物5g，加水1升灭菌后使用)，于37℃下170转/分钟摇床中培养1小时后，5000转/分钟离心1分钟。将沉淀涂在含有抗生素活性的LB琼脂糖(氯化钠10g，蛋白胨10g；酵母提取物5g，琼脂粉3g，加水1升灭菌后使用)板上，待生长16小时后取出，挑单克隆菌落。
3、CRISPR-CAS9慢病毒克隆载体测序，将上述挑选的单克隆菌落加入LB培养基中进行进行扩大培养，同时加入氨苄青霉素抗生素(100g/ml)进行抗性筛选，37℃下220转/分钟摇床中培养12小时后，抽提质粒测序。
测序结果显示重组克隆质粒中，以上两个正义链模板已经插入到载体中：
重组克隆构建成功。
实施例3(普通克隆重组法构建的具有嘌呤霉素抗性的CRISPR-CAS9慢病毒载体基因敲除，以PKA基因为例)
1、大鼠PKA基因sgRNA设计：
(1)设计获得PKA基因的正、反义链引物序列：
sgRNA1F：AACGCCGCCGCCGCCAAGAAGTTTT SEQ ID No.11
sgRNA1R：TTCTTGGCGGCGGCGGCGTTCGGTG SEQ ID No.12
sgRNA2F：CCTCCCAATCCGCCGTAAGTGTTTT SEQ ID No.13
sgRNA2R：ACTTACGGCGGATTGGGAGGCGGTG SEQ ID No.14
sgRNA3F：CGATCTGCGCCGCGTAGAAAGTTTT SEQ ID No.15
sgRNA3R：TTTCTACGCGGCGCAGATCGCGGTG SEQ ID No.16
(2)试剂盒所述的线性化具有嘌呤霉素抗性的CRISPR-CAS9慢病毒载体；
(3)T4连接酶和T4连接酶缓冲液；
(4)高转化活性Stbl3感受态；
2、具有嘌呤霉素抗性的CRISPR-CAS9慢病毒克隆载体的重组
(1)将正义链和反义链退火形成双链：取等量的正义链和反义链混合(终浓度为10μM)。程序如下：37℃，30分钟；95℃，5分钟；94℃，12sec，-l℃/循环，70个循环；最终为24℃，具体反应的样品体积和浓度如下。
1μl正义链模板(100mM)
1μl反义链模板(100mM)
1μl T4连接酶缓冲液
加入灭菌去离子水至总体积10μl。
反应完成后，在10μl中加入2ml灭菌去离子水。
(2)将上述得到的正义链和反义链混合物和试剂盒中的线性化CRISPR-CAS9载体加入试剂盒中的T4连接酶连接，连接20分钟。具体方法是：
1μl线性化的具有嘌呤霉素抗性的CRISPR-CAS9载体
1μl正义链和反义链混合物
1μl T4连接酶缓冲液
1μl T4连接酶
加入灭菌去离子水至总体积10μl。
(3)将本试剂盒中高转化活性的Stbl3感受态在冰上解冻，将上述11μl的正义链和反义链混合物加入感受态细胞并摇匀，冰上放置约30分钟。水浴42℃热激90秒后，加入1毫升LB培养基(氯化钠10g，蛋白胨10g；酵母提取物5g，加水1升灭菌后使用)，于37℃下170转/分钟摇床中培养1小时后，5000转/分钟离心1分钟。将沉淀涂在含有抗生素活性的LB琼脂糖(氯化钠10g，蛋白胨10g；酵母提取物5g，琼脂粉3g，加水1升灭菌后使用)板上，待生长16小时后取出，挑单克隆菌落。
3、CRISPR-CAS9慢病毒克隆载体测序，将上述挑选的单克隆菌落加入LB培养基中进行进行扩大培养，同时加入氨苄青霉素抗生素(100g/ml)进行抗性筛选，37℃下220转/分钟摇床中培养12小时后，抽提质粒测序。
测序结果显示重组克隆质粒中，以上两个正义链模板已经插入到载体中：
重组克隆1：AAACGCAACGCATTACTTTGGAGTC SEQ ID No.17
重组克隆2：CACCGCACGTGGATCAGCGTCGCCA SEQ ID No.18
重组克隆构建成功。
SEQUENCE LISTING
<110> 南方医科大学
<120> 一种基于线性化CRISPR-CAS9慢病毒载体基因敲除试剂盒及其应用
<160> 18
<170> PatentIn version 3.3
<210> 1
<211> 25
<212> DNA
<213> sgRNA1F
<400> 1
aaacgcaacg cattactttg gagtc 25
<210> 2
<211> 25
<212> DNA
<213> sgRNA1R
<400> 2
caccgcacgt ggatcagcgt cgcca 25
<210> 3
<211> 25
<212> DNA
<213> sgRNA2F
<400> 3
caccgcacgt ggatcagcgt cgcca 25
<210> 4
<211> 25
<212> DNA
<213> sgRNA2R
<400> 4
aaactggcga cgctgatcca cgtgc 25
<210> 5
<211> 25
<212> DNA
<213> sgRNA1F
<400> 5
tcgtgagcga cccggacacc gtttt 25
<210> 6
<211> 25
<212> DNA
<213> sgRNA1R
<400> 6
ggtgtccggg tcgctcacga cggtg 25
<210> 7
<211> 25
<212> DNA
<213> sgRNA2F
<400> 7
cagcttccac gtgttcgacc gtttt 25
<210> 8
<211> 25
<212> DNA
<213> sgRNA2R
<400> 8
ggtcgaacac gtggaagctg cggtg 25
<210> 9
<211> 25
<212> DNA
<213> sgRNA3F
<400> 9
ggagtcaatg agggcggtcg gtttt 25
<210> 10
<211> 25
<212> DNA
<213> sgRNA3R
<400> 10
cgaccgccct cattgactcc cggtg 25
<210> 11
<211> 25
<212> DNA
<213> sgRNA1F
<400> 11
aacgccgccg ccgccaagaa gtttt 25
<210> 12
<211> 25
<212> DNA
<213> sgRNA1R
<400> 12
ttcttggcgg cggcggcgtt cggtg 25
<210> 13
<211> 25
<212> DNA
<213> sgRNA2F
<400> 13
cctcccaatc cgccgtaagt gtttt 25
<210> 14
<211> 25
<212> DNA
<213> sgRNA2R
<400> 14
acttacggcg gattgggagg cggtg 25
<210> 15
<211> 25
<212> DNA
<213> sgRNA3F
<400> 15
cgatctgcgc cgcgtagaaa gtttt 25
<210> 16
<211> 25
<212> DNA
<213> sgRNA3R
<400> 16
tttctacgcg gcgcagatcg cggtg 25
<210> 17
<211> 25
<212> DNA
<213> 重组克隆1
<400> 17
aaacgcaacg cattactttg gagtc 25
<210> 18
<211> 25
<212> DNA
<213> 重组克隆2
<400> 18
caccgcacgt ggatcagcgt cgcca 25
Claims (7)
1.一种基于CRISPR-CAS9的基因重组试剂盒，该试剂盒包括CRISPR-CAS9载体、T4连接酶及感受态细胞，其特征在于所述的CRISPR-CAS9载体为线性CRISPR-CAS9载体，优选为慢病毒载体、质粒载体或腺病毒载体。
2.根据权利要求1所述的基因重组试剂盒，所述的线性CRISPR-CAS9载体通过以BsmBI酶酶切CRISPR-CAS9慢病毒环状载体获得，并通过琼脂糖凝胶进行回收；其中所述酶切的反应液中每50μl中CRISPR-CAS9慢病毒环状载体为2μg，BsmBI酶50U。
3.根据权利要求1所述的基因重组试剂盒，所述的感受态细胞为Stbl3感受态细胞，优选为经过氯化钙处理的Stbl3感受态细胞。
4.根据权利要求1所述的基因重组试剂盒，其中还包含T4连接酶缓冲液，T4连接酶的剂量为每10ng线性化CRISPR-CAS9载体配20U T4连接酶。
5.根据权利要求1所述的基因重组试剂盒，其中还包含目的基因的sgRNA寡核苷酸的正义链模板和反义链模板，正义链模板的5’端添加CACC；反义链模板的5’端添加AAAC。
6.根据权利要求1所述的基因重组试剂盒，该试剂盒包括10ng线性CRISPR-CAS9慢病毒载体、1-2μl 20U/μl T4连接酶、1μl T4连接酶缓冲液及Stbl3感受态细胞。
7.一种以权利要求1-6任一项所述的基因重组试剂盒进行基因重组的方法，其包括如下步骤：
1)将sgRNA寡核苷酸的正义链模板和反义链模板退火形成双链；
2)将步骤1)得到的正义链和反义链混合物和基因重组试剂盒中的线性化CRISPR-CAS9载体与T4连接酶进行连接反应，获得连接目的基因的sgRNA寡核苷酸的载体；
3)将连接目的基因的sgRNA寡核苷酸的载体以感受态细胞进行培养，并通过培养基培养获得单克隆菌落。
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