CN107034229A - 一种植物中高效筛选CRISPR/CAS9基因编辑系统候选sgRNA系统及应用 - Google Patents
一种植物中高效筛选CRISPR/CAS9基因编辑系统候选sgRNA系统及应用 Download PDFInfo
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Abstract
本发明公开了一种CRISPR/CAS9系统候选sgRNA靶位点高通量筛选系统及其应用，所述测试系统包括：(1)用于同时表达多个候选sgRNA的质粒；(2)用于表达CAS9的质粒；(3)利用拟南芥及水稻原生质体瞬时表达系统快速高通量鉴定候选sgRNA靶位点切割效率。CRISPR/CAS9系统在敲除或编辑基因之前，选择可以高效切割的靶序列至关重要，短时间内验证多个候选sgRNA靶位点的切割效率，从而选择最佳的sgRNA提高敲除的成功效率，不仅可以降低工作成本，还可以提高工作效率。
Description
技术领域
本发明涉及一种植物高效筛选CRISPR/CAS9基因编辑系统候选sgRNA系统及应用，可利用拟南芥(双子叶植物)、水稻(单子叶植物)瞬时表达系统，快速高效筛选可有效用于编辑植物基因组的sgRNA靶位点。
技术背景
基因组编辑技术是人类改造生物基因组的重要技术，在农业、医学及科研等领域有巨大的应用价值。CRISPR/CAS9因其简单高效成为目前最常用的基因组编辑系统。它是由一种来自细菌及古细菌中降解入侵的病毒或其他外源的DNA序列的免疫机制改造而来。它包括三个要素：CAS9蛋白、cr(CRISPR-derived RNA)RNA以及tracr(trans-activatingcrRNA)RNA。在CRISPR/CAS9系统中，crRNA通过碱基配对与tracrRNA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。而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。CAS9蛋白含有两个核酸酶结构域RuvC和HNH，在CRISPR/CAS9复合物通过sgRNA与靶DNA配对后RuvC及HNH结构域各自切割靶DNA的一条链，从而形成双链DNA断裂(DSB,double strand break)。DSB发生后由细胞内的两条修复途径进行修复，即：保真性高的同源介导修复(Homology-directed repair,HDR)及低保真性的非同源末端连接途径(NHEJ，Non-homologous end joining)。保真性高的同源介导修复途径在有同源DNA模板存在的情况下可以实现基因组的精确编辑，而低保真性的非同源末端连接途径的修复可以产生碱基的删除、插入及突变，从而导致基因突变或移码，从而使基因失活，达到敲除基因的目的。所以CRISPR/CAS9系统在基因组编辑领域有巨大的应用潜力，且已在短时间内得以大量应用。
CRISPR/CAS9基因编辑系统的工作效率主要取决于sgRNA的靶向切割效率,在CRISPR/CAS9系统应用中,对靶的识别能力差、与目的的靶位亲和力低或的切割效率低下都会降低CRISPR/CAS9的工作效率,限制了CRISPR/CAS9的实际应用，所以快速测试候选靶向切割效率及随后的基因编辑效率,将大大提高CRISPR/CAS9的使用效率并降低成本。
基因组编辑技术在农业方面有重要的应用价值。传统育种方式通过多年杂交和大量的表型筛选将优良基因聚合到一起，这种方式时间长、效率低、工作量大。所以通过基因组编辑技术对特定基因进行敲除或编辑可以快速将聚合优良基因淘汰不良基因，从而大幅提高育种效率。CRISPR/CAS9因其简单高效的特性在植物基因组编辑操作中有巨大优势，在实际操作用，sgRNA靶位点的选择对基因组编辑效率有重要作用。目前的sgRNA靶位点的选择有两种方式，一种是通过计算机预测来进行候选靶位点的筛选，如：张锋实验室的sgRNA靶点在线设计网站http://crispr.mit.edu/。另一种是在报告基因中加入靶位点，通过检测报告基因的表达情况对候选sgRNA靶位点的切割效率进行检测。第一种方法仅是计算机预测的结果，并未真实验证，依然有可能出现sgRNA靶位点切割效率不高甚至无法切割的情况。后一种方法只能一次检测一个候选sgRNA靶位点，无法实现高通量的筛选，且对报告基因的检测是间接性检测，并无法直接体现sgRNA靶位点的真实切割情况。所以在植物基因组编辑领域急需一种高通量快速验证候选sgRNA靶位点切割能力的检测系统。
发明内容
本发明的目的是构建了一种在两种模式植物拟南芥(双子叶植物)及水稻(单子叶植物)的瞬时转化系统中同时验证多个候选sgRNA靶位点切割效率系统，为高效筛选候选sgRNA提供可靠的核心技术基础。
本发明的目的是通过以下技术方案实现的：
本发明提供一种多个候选sgRNA靶位点快速测试系统，所述测试系统包括：(1)用于拟南芥(双子叶植物)中同时表达多个候选sgRNA的质粒；(2)用于水稻(单子叶植物)中同时表达多个候选sgRNA的质粒；(2)用于表达的CAS9质粒
进一步，本发明中的候选sgRNA高通量筛选分别在拟南芥及水稻的原生质体瞬时转化系统中实现。
进一步，本发明中用于拟南芥中同时表达多个候选sgRNA的质粒pDgRNA(图1)的构建过程如下：将常用克隆载体pUC19骨架上的Bsa I酶切位点上设计一对反向互补的引物并将酶切位点突变为序列(GGGACC)，用这对反向扩增引物，用高保真酶扩增整个载体，用DpnI消化模板质粒，将产物转化DH5α大肠杆菌并提取质粒，通过测序确定pUC19骨架上的Bsa I酶切位点被突变。在由基因合成的五个串联sgRNA表达框两端加入EcoR I及Hind III酶切位点，并用常规分子克隆方法将串联的sgRNA表达框连到改造后的pUC19载体。拟南芥系统中表达的sgRNA由拟南芥中的AtU3b启动子驱动并在转录起始位点旁引入一对方向相反的Bsa I的酶切位点，可以利用Golden Gate方法将候选靶序列连入相应位点。
进一步，本发明中用于水稻中同时表达多个候选sgRNA的质粒pMgRNA(图2)的构建过程如下：在由基因合成的五个串联sgRNA表达框两端加入EcoR I及Hind III酶切位点，并用常规分子克隆方法将串联的sgRNA表达框连到上述改造后的pUC19载体。水稻系统中表达的sgRNA由水稻中的OsU6b启动子驱动并在转录起始位点旁引入一对方向相反的Bsa I的酶切位点，可以利用Golden Gate方法将候选靶序列连入相应位点。
进一步，本发明中用于表达CAS9蛋白的载体构建方法如下：用infusion同源重组方法将由基因合成的CAS9表达框连入pUC19的EcoR I酶切位点处。即pUCCAS9质粒。
进一步，本发明中的sgRNA靶位点切割效率检测系统操作时将候选靶位点序列利用引物进行合成，并形成与要插入的sgRNA表达框的中相应位点粘性末端互补，采用边切边连的方法利用Bsa I和T4 DNA连接酶同时进行酶切和连接。通过测序确定重组质粒序列是否正确。
进一步，采用贝瑞丽公司的植物原生质体分离试剂盒分离拟南芥及水稻的原生质体。将上述表达sgRNA的重组质粒及CAS9表达质粒同时转入拟南芥及水稻原生质体中。后续实验通过测序确定不同sgRNA靶位点的切割效率的差异。
本发明的有益技术效果：
(1)本发明提供一个易于构建的多个sgRNA同时表达的载体质粒，采用Bsa I和T4DNA连接酶边切边连的方法可以高效快速构建好重组载体。
(2)本发明中采用多个sgRNA同时表达的方式来比较不同sgRNA靶位点在同一次实验中的切割效率的差异。
(3)本发明中采用同时检测多个sgRNA的切割效率的方式可以大大提高检测效率，降低成本。
(4)与现有技术相比，本发明所述构建方法方便，检测通量高，成本低，适合于植物等。
附图说明
图1为载体质粒pDgRNA示意图
图2为载体质粒pMgRNA示意图
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合附图及实施例对本发明进行进一步详细描述。应当理解，此处所描述的具体实施例仅用于解释本发明并不用于限定本发明。相反,本发明涵盖任何由权利要求定义的在本发明的精髓和范围上做的替代、修改、等效方法以及方案。进一步,为了使公众对本发明有更好的了解,在下文对本发明的细节描述中,详尽描述了一些特定的细节部分。对本领域技术人员来说没有这些细节部分的描述也可以完全理解本发明。
实验例1：筛选拟南芥Rubisco small subunit基因候选sgRNA
利用华中农业大学的CRISPR/CAS9靶位点设计网站http://cbi.hzau.edu.cn/对Rubisco small subunit基因预测候选sgRNA靶位点选择5个高分靶位点，将它们采用边切边连的方法将这5个靶位点连入pDgRNA载体，并通过测序确认重组质粒序列。靶点序列如下：
sgRNA靶序列1：GTCGTTGTTAGCCTTGCGGGTGG
sgRNA靶序列2：CGTGAGCACGGTAACTCACCCGG
sgRNA靶序列3：ATAGAATATGTCTCGCAAACCGG
sgRNA靶序列4：GGAGTCGGTGCAACCGAACAAGG
sgRNA靶序列5：CGGAATCGGTAAGGTCAGGAAGG
用贝瑞利生物科技有限公司的植物原生质体提取试剂盒分离拟南芥原生质体。将上述质粒及CAS9表达载体pUCCAS9同时转入拟南芥原生质体。待转化后的原生质体培养16小时后提取基因组DNA。用Rubisco small subunit基因特异性引物扩增后，连接克隆载体，随机挑选50个单克隆，对编辑区域测序，分析测序结果，将5个靶位点均无编辑的克隆排除，计算各位点的编辑频率比为：12:3:39:1:22所以sgRNA靶序列3为切割效率较高的靶点。
实验例2：筛选水稻PAPST1基因候选sgRNA
利用华中农业大学的CRISPR/CAS9靶位点设计网站水稻PAPST1基因候选sgRNA靶位点选择5个高分靶位点，将它们采用边切边连的方法将这5个靶位点连入pMgRNA载体，并通过测序确认重组质粒序列。靶点序列如下：
sgRNA靶序列1：CCGCATAGTTCCTTACAGTGCGG
sgRNA靶序列2：CCGCACTGTAAGGAACTATGCGG
sgRNA靶序列3：ACATCATCAGAGTTACCTCGAGG
sgRNA靶序列4：CATGAATCAAGTCTTCGGACTGG
sgRNA靶序列5：GCATCCAAAACCGTGTTGTAGGG
用贝瑞利生物科技有限公司的植物原生质体提取试剂盒分离水稻叶鞘原生质体。将上述质粒及CAS9表达载体pUCCAS9同时转入水稻原生质体。待转化后的原生质体培养16小时后提取基因组DNA。用水稻PAPST1基因特异性引物扩增后，连接克隆载体，随机挑选50个单克隆，对编辑区域测序，分析测序结果，将5个靶位点均无编辑的克隆排除，计算各位点的编辑频率比为：2:13:26:18:43所以sgRNA靶序列5为切割效率较高的靶点。
Claims (4)
1.一种CRISPR/CAS9系统候选sgRNA切割效率筛选系统，其特征在于测试系统包括：(1)用于同时表达多个sgRNA的质粒；(2)用于表达CAS9的质粒。
2.如权利要求1所述CRISPR/CAS9工作效率高通量测试系统，其特征在于能够在同一次实验中同时表达多个候选sgRNA。
3.如权利要求1所述其特征在于利用拟南芥及水稻原生质体瞬时表达系统进行高通量sgRNA切割效率筛选。
4.如权利要求1所述其特征在于构建多sgRNA表达质粒时利用Bsa I为酶切位点切割质粒并利用T4DNA连接酶连接。
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