CN108642055B - 能有效编辑猪miR-17-92基因簇的sgRNA - Google Patents
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Abstract
本发明提供一条能有效编辑猪miR‑17‑92基因簇的sgRNA，以能特异识别猪miR‑17‑92基因簇的sgRNA为前提，利用CRISPR/Cas9和RNAi技术，成功将shRNA定点整合到猪肾细胞PK‑15‑EGFP‑KI细胞，并筛选出敲低了EGFP基因表达的阳性细胞克隆；利用上述sgRNA将shRNA定点整合到猪胎儿成纤维细胞，成功筛选出shRNA定点整合的阳性细胞系，并检测到shRNA的稳定转录和定点整合事件，该细胞系的制备过程中，shRNA在猪miR‑17‑92基因簇的启动子的作用下稳定转录和有效表达，没有引入任何的外源的启动子基因及正负筛选标记基因，增加了转基因猪的安全性，对去除转基因猪的生物安全隐患具有重要意义。
Description
技术领域
本发明公开了一条能有效编辑猪miR-17-92基因簇的sgRNA及其应用，同时还公开了与该sgRNA相关的更加安全的外源基因的定点策略及可行性分析，属于生物技术领域。
技术背景
CRISPR/Cas9最早在细菌和古细菌中被发现，是生物体为应对病毒和质粒不断攻击而演化来的获得性免疫防御机制。CRISPR/Cas9 系统的工作原理是crRNA（CRISPR-derived RNA ）通过碱基配对与tracrRNA（trans-activating RNA）结合形成 tracrRNA/crRNA 复合物，此复合物引导核酸酶Cas9蛋白在与crRNA 配对的序列靶位点剪切双链DNA。通过人工设计这两种RNA，可以改造形成具有引导作用的sgRNA（single-guide RNA ），足以引导 Cas9对DNA的定点切割。CRISPR/Cas9是一种精确且万能基因编辑工具，可以实现基因组的精确编辑，如条件性基因敲除、基因敲入、基因替换、点突变等。CRISPR/Cas9 技术弥补了传统基因编辑技术的诸多不足，因其效率高、速度快、生殖系转移能力强及简单经济等特点迅速获得青睐，在基因编辑领域和动物模型构建的应用前景非常广阔。
RNA干扰（RNAi）是一种由内源性或外源性双链RNA分子引发的转录后基因沉默现象，是生物体本身存在的对抗侵入的病毒、转基因等外源基因，抑制其循环复制从而减弱其基因毒性作用的一种保护自身的天然机制。小干扰RNA（Small interfering RNA, siRNA）有时称为短干扰RNA（short interfering RNA, shRNA），是一个长20到25个核苷酸的双股RNA。利用dicer酶将较长的双股RNA或小发夹RNA（small hairpin RNA）切成siRNA。shRNA或siRNA可经由多种不同转染（transfection）技术导入细胞内，并对特定基因产生具专一性的敲弱（knock down）效果。由于其作用的特异性、高效性，已广泛应用于抗病毒等方面的研究。
由于猪与人的各器官的结构与血液生理生化指标相似，转基因猪成为更好的动物模型。目前，对体细胞的基因修饰主要依赖将外源基因随机插入猪基因组的方法，导致外源基因在猪基因组中的整合位点和拷贝数不可控，进而使外源基因猪体内表达不稳定，引发转基因猪个体之间表型不均一等问题。又因为传统转基因猪的制备过程中对外源启动子基因及正负筛选标记基因的依赖性，进一步增加了转基因猪的安全评价等问题。这些因素限制了转基因猪的培育与应用前景。
发明内容
本发明的目的是提供一条能有效编辑猪miR-17-92基因簇的sgRNA，该sgRNA能用于特异识别和靶向编辑猪miR-17-92基因簇及miR-17-92基因簇定点整合转基因猪的制备。
本发明所提供了一种能有效编辑猪miR-17-92基因簇的sgRNA，其特征在于：该sgRNA的RNA序列如：SEQ ID 1所示，互补链的RNA序列如：SEQ ID 2所示。
本发明能有效编辑猪miR-17-92基因簇的sgRNA的DNA序列，其特征在于：该sgRNA的DNA序列如SEQ ID 3所示，互补链的DNA序列如：SEQ ID 4所示。
本发明所述的能有效编辑猪miR-17-92基因簇的sgRNA的制备方法，包括以下步骤：
1）在基因库中调出猪miR-17-92基因簇的序列，根据PAM序列（NGG）选择用于基因编辑的sgRNA靶向的区域；
2）根据靶位点的序列，设计并合成对应的引物序列；
3）引物退火形成寡聚核苷二聚体（oligoduplex）；
4）将寡核苷酸二聚体连接到相应的质粒载体中，即可获得sgRNA的表达载体；
本发明所述的能有效编辑猪miR-17-92基因簇的sgRNA在猪miR-17-92位点编辑的应用。
本发明所述的能有效编辑猪miR-17-92基因簇的sgRNA在猪miR-17-92位点编辑的应用，其特征在于：能有效编辑猪miR-17-92基因簇的sgRNA在特异识别和靶向编辑猪miR-17-92基因簇中的应用以及在制备miR-17-92基因簇定点整合转基因猪中的应用。
本发明的积极效果在于：
以猪基因组中的一个能特异识别猪miR-17-92基因簇的sgRNA为前提，利用CRISPR/Cas9和RNAi技术，成功将shRNA（针对EGFP基因）定点整合到猪肾细胞PK-15-EGFP-KI细胞，并筛选出敲低了EGFP基因表达的阳性细胞克隆，结果显示该阳性细胞克隆中shRNA能稳定转录且有效抑制EGFP基因的表达；另外，我们进一步利用上述sgRNA将shRNA定点整合到猪胎儿成纤维细胞，成功筛选出shRNA定点整合的阳性细胞系，并检测到shRNA的稳定转录和定点整合事件，该细胞系的制备过程中，shRNA在猪miR-17-92基因簇的启动子的作用下稳定转录和有效表达，没有引入任何的外源的启动子基因及正负筛选标记基因，因此该位点有成为制备转基因猪安全位点的可能和潜力，这也大大的增加了转基因猪的安全性，对去除转基因猪的生物安全隐患具有重要意义。
附图说明：
图1：用于评估3条不同sgRNA切割效率的测序峰图；
图2：shRNA定点打靶质粒载体示意图；
图3：shRNA RT-PCR鉴定电泳图和测序峰图；
图4：shRNA定点整合猪PK-15-EGFP-KI细胞阳性克隆的荧光显微镜图；
图5：shRNA RT-PCR和定点整合猪胎儿成纤维细胞的鉴定电泳图。
具体实施方式
通过以下实施例进一步举例描述本发明，并不以任何方式限制本发明，在不背离本发明的技术解决方案的前提下，对本发明所作的本领域普通技术人员容易实现的任何改动或改变都将落入本发明的权利要求范围之内。
实施例1
1-1、sgRNA序列的设计及PX330表达载体的构建
设计并合成了3条针对猪miR-17-92位点的sgRNA序列。上述设计完成的sgRNA序列合成；6条单链的sgRNA的DNA序列分别经过退火后形成3条靶向猪miR-17-92位点3’-UTR不同位点的sgRNA的寡核苷酸链；然后将该寡聚核苷酸连连入PX330质粒载体。
这3条sgRNA的序列及其作用位点的序列分别为：
SgRNA-1序列：5-atgattctgtaccacttgtg-3
SgRNA-1作用位点的序列：5-CACAAGTGGTACAGAATCAT-3
SgRNA-2序列：5-GCTGTATTGTCAGATTTATC-3
SgRNA-2作用位点的序列：5- GATAAATCTGACAATACAGC-3
SgRNA-3序列：5-attctgtaccacttgtgagg-3
SgRNA-3作用位点的序列：5-CCTCACAAGTGGTACAGAAT-3
其中，本发明所涉及的
sgRNA的RNA序列为：5-aUgaUUcUgUaccacUUgUg-3 （SEQ ID NO.1）
sgRNA的RNA序列的互补序列为：5-CACAAGUGGUACAGAAUCAU-3 （SEQ ID NO.2）
其中，本发明所涉及的
sgRNA的DNA序列为：5-atgattctgtaccacttgtg-3 （SEQ ID NO.3）
sgRNA的DNA序列的互补序列为：5-CACAAGTGGTACAGAATCAT-3 （SEQ ID NO.4）。
1-2、高效sgRNA的评估和筛选
通过对构建的3种PX330-sgRNA表达载体测序验证后，提取目的质粒和进行乙醇沉淀，将纯化后一定浓度的三种PX330-sgRNA表达载体，通过电穿孔转染的方式引入到猪PK-15-EGFP-KI细胞系中，转染72小时后，提取各组细胞的基因组，然后用特异性的检测突变效率的引物进行PCR反应，将所获得的PCR产物送去测序通过对测序峰图的分析初步评估个sgRNA的切割效率, 同时将剩下的PCR产物用于连T载体或通过T7E1分析来精确的评估各sgRNA对靶基因的切割效率（参见图1，用于评估3条不同sgRNA切割效率的测序峰图）。
实施例2
shRNA定点整合打靶载体的构建
根据筛选出的高效sgRNA设计并构建与该sgRNA配套的shRNA定点整合打靶载体（pLB-shRNA-KI-Donor），该打靶载体的主要原件依次为：上游同源臂、shRNA-EGFP基因、下游同源臂及原核表达的骨架载体。该shRNA定点整合打靶质粒与筛选出的sgRNA共同作用可以对猪miR-17-92位点进行特异的基因改造，然后再结合荧光显微镜与PCR的方法可以很方便的分析外源基因在该sgRNA识别位点整合并表达的可行性（图2，shRNA定点打靶质粒载体示意图）。
实施例3
PX330质粒与pLB-shRNA-KI-Donor质粒的共转染
复苏PK-15-EGFP-KI细胞，待接近长满时，用DPBS洗2~3遍后，弃上清，加入电转染缓冲液，然后将PX330质粒与pLB-shRNA-KI-Donor质粒按比例加入到细胞和缓冲液中，用移液器轻轻混匀后，轻轻的将混合液移入电极杯中，将电转杯放到电穿孔仪器上进行电击操作。电击完成后，电转杯静置10分钟后，将电转杯中的混合液转入细胞培养皿中。最后将该细胞培养皿置于37℃二氧化碳培养箱中培养。培养12小时后，换液。
实施例4
shRNA定点整合猪PK-15-EGFP-KI细胞克隆的挑取与观察
电转染72h后，使用胰酶消化一部分细胞提取总RNA，使用设计的特异性引物反转录成cDNA，通过RT-PCR和测序的方法验证shRNA是否正常转录；另一部分细胞通过极限稀释的方法将猪PK-15-EGFP-KI细胞铺到100mm细胞培养皿中，2~3天更换一次细胞培养液。8~10天后待细胞克隆长成后，在荧光显微镜下将绿光显著减弱的细胞克隆统一做上标记，然后将这些标记过的克隆挑取入24孔细胞培养板中接着培养。2~3天后，待24孔板中的细胞长至一定得汇合度，根据荧光显微镜观察细胞克隆的发光情况，将绿色荧光显著减弱的细胞克隆进行标记同时分出部分克隆的细胞，将这些细胞用NP40裂解液裂解后再通过PCR和测序的方法进一步验证shRNA转录和定点整合事件。（参见图3，shRNA RT-PCR鉴定电泳图（A）和测序峰图（B）；图4，shRNA定点整合猪PK-15-EGFP-KI细胞阳性克隆的荧光显微镜图）
实施例5
shRNA定点整合猪胎儿成纤维细胞克隆的挑取与鉴定
复苏猪原代胎儿成纤维细胞，使用F3代的猪胎儿成纤维细胞和上述质粒进行电穿孔转染，电击完成后静置10分钟，将电转杯中的混合液转入细胞培养皿置于37℃二氧化碳培养箱中培养。培养12小时后，换液。电转染72h后，使用胰酶消化一部分细胞提取总RNA，使用设计的特异性引物反转录成cDNA，通过RT-PCR和测序的方法验证shRNA是否正常转录；另一部分细胞通过极限稀释的方法将猪胎儿成纤维细胞铺到100mm细胞培养皿中，2~3天更换一次细胞培养液。9~10天后待细胞克隆长成后，将标记过的克隆挑取入24孔细胞培养板中接着培养。2~3天后，待24孔板中的细胞长至一定得汇合度，对细胞进行传代同时分出部分克隆的细胞，将这些细胞用NP40裂解液裂解后再通过PCR和测序的方法进一步验证shRNA转录和定点整合事件（参见图5，shRNA RT-PCR（A）和定点整合猪胎儿成纤维细胞（B）的鉴定电泳图）。
序列表
<110> 吉林大学
<120> 能有效编辑猪miR-17-92基因簇的sgRNA
<160> 6
<170> SIPOSequenceListing 1.0
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<212> RNA
<213> 人工序列(sgRNA)
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<212> RNA
<213> 人工序列(sgRNA)
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gcuguauugu cagauuuauc 20
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<212> RNA
<213> 人工序列(sgRNA)
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auucuguacc acuugugagg 20
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<213> 人工序列(sgRNA)
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atgattctgt accacttgtg 20
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<213> 人工序列(sgRNA)
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attctgtacc acttgtgagg 20
Claims (5)
1.一种能有效编辑猪miR-17-92基因簇的sgRNA，其特征在于：
该sgRNA的RNA序列如：SEQ ID No .1所示。
2.如权利要求1所述的能有效编辑猪miR-17-92基因簇的sgRNA的制备方法，包括以下步骤：
1）在基因库中调出猪miR-17-92基因簇的序列，根据PAM序列选择用于基因编辑的sgRNA靶向的区域；
2）根据靶位点的序列，设计并合成对应的引物序列；
3）引物退火形成寡聚核苷二聚体；
4）将寡核苷酸二聚体连接到相应的质粒载体中，即可获得sgRNA的表达载体。
3.如权利要求1所述的能有效编辑猪miR-17-92基因簇的sgRNA在猪miR-17-92位点编辑中的应用；
其特征在于：能有效编辑猪miR-17-92基因簇的sgRNA在特异识别和靶向编辑猪miR-17-92基因簇中的应用以及在制备miR-17-92基因簇定点整合转基因猪中的应用。
4.如权利要求1所述的能有效编辑猪miR-17-92基因簇的sgRNA在特异识别和靶向编辑猪miR-17-92基因簇中的应用。
5.如权利要求1所述的能有效编辑猪miR-17-92基因簇的sgRNA在制备miR-17-92基因簇定点整合转基因猪中的应用。
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| US9388430B2 (en) | 2013-09-06 | 2016-07-12 | President And Fellows Of Harvard College | Cas9-recombinase fusion proteins and uses thereof |
| US9228207B2 (en) | 2013-09-06 | 2016-01-05 | President And Fellows Of Harvard College | Switchable gRNAs comprising aptamers |
| US9737604B2 (en) | 2013-09-06 | 2017-08-22 | President And Fellows Of Harvard College | Use of cationic lipids to deliver CAS9 |
| US11053481B2 (en) | 2013-12-12 | 2021-07-06 | President And Fellows Of Harvard College | Fusions of Cas9 domains and nucleic acid-editing domains |
| EP3177718B1 (en) | 2014-07-30 | 2022-03-16 | President and Fellows of Harvard College | Cas9 proteins including ligand-dependent inteins |
| EP3365356B1 (en) | 2015-10-23 | 2023-06-28 | President and Fellows of Harvard College | Nucleobase editors and uses thereof |
| CN110214183A (zh) | 2016-08-03 | 2019-09-06 | 哈佛大学的校长及成员们 | 腺苷核碱基编辑器及其用途 |
| US11661590B2 (en) | 2016-08-09 | 2023-05-30 | President And Fellows Of Harvard College | Programmable CAS9-recombinase fusion proteins and uses thereof |
| WO2018039438A1 (en) | 2016-08-24 | 2018-03-01 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
| CA3039928A1 (en) | 2016-10-14 | 2018-04-19 | President And Fellows Of Harvard College | Aav delivery of nucleobase editors |
| WO2018119359A1 (en) | 2016-12-23 | 2018-06-28 | President And Fellows Of Harvard College | Editing of ccr5 receptor gene to protect against hiv infection |
| US11898179B2 (en) | 2017-03-09 | 2024-02-13 | President And Fellows Of Harvard College | Suppression of pain by gene editing |
| CN110914310A (zh) | 2017-03-10 | 2020-03-24 | 哈佛大学的校长及成员们 | 胞嘧啶至鸟嘌呤碱基编辑器 |
| IL269458B2 (en) | 2017-03-23 | 2024-02-01 | Harvard College | Nucleic base editors that include nucleic acid programmable DNA binding proteins |
| US11560566B2 (en) | 2017-05-12 | 2023-01-24 | President And Fellows Of Harvard College | Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation |
| US11732274B2 (en) | 2017-07-28 | 2023-08-22 | President And Fellows Of Harvard College | Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE) |
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