CN106906242A - 一种提高CRIPSR/Cas9靶向敲除基因产生非同源性末端接合效率的方法 - Google Patents
一种提高CRIPSR/Cas9靶向敲除基因产生非同源性末端接合效率的方法 Download PDFInfo
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Abstract
本发明属于分子生物学与生物医学技术领域，本发明涉及一种提高CRIPSR/Cas9靶向敲除基因NHEJ效率的方法。具体来说，本发明设计筛选出高效干扰pten基因表达的小分子干扰RNA，使用CRISPR/Cas9系统靶向敲除基因时，共转染该小分子RNA，可以有效的提高靶基因的NHEJ效率。在细胞多个靶点验证，均有明显提高，该方法成本低、操作简单、效率高，对于CRISPR/Cas9技术的效率和应用具有很好的促进作用。
Description
技术领域
本发明属于分子生物学与生物医学技术领域，具体涉及一种提高CRIPSR/Cas9靶向敲除基因非同源性末端接合(Non-homologous end joining，简称NHEJ)效率的方法。
背景技术
CRISPR/Cas系统是从细菌和古生菌对抗外来病毒或质粒的适应性免疫系统发展而来，包括三种不同的类型，其中 Type Ⅱ型的 CRISPR/Cas系统的 DNA 内切酶Cas9 只有一个亚基，结构最为简单，所以应用也最广泛。除了Cas9 蛋白外，该系统还包括两条短的CRISPR RNAs（crRNAs）和 trans-activating crRNAs（tracrRNA）。成熟的crRNA-tracrRNA复合体可以通过碱基互补配对指导Cas9 蛋白到靶序列上，并在 PAM（protospaceradjacent motif）附近特异性剪切 DNA 双链，形成 DSB（double strand break）。DSB 可以通过两种途径被修复，一种是非同源性末端接合（Non-Homologous End Joining NHEJ）DNA修复方式，另一种是同源重组修复（ Homology Directed Repair HDR) 方式。NHEJ 修复方式可能产生碱基的插入或缺失，从而产生移码突变，或者也可能突变成终止密码子，这些突变形式都可以改变目的基因的开放阅读框；HDR 方式需要一段与被剪切片段同源的模板片段来修复DSB，这种修复方式可以将被用来作为模板的同源片段的序列复制到目的基因中，所以可以利用这种修复方式将特定的基因片段引入到目的基因中。
Cas9有时较差的切割效率可能与DNA是如何修复的相关联，这是因为DNA修复机制---基本的管家酶修复DNA中的任何可能导致致命性突变的断裂或缺失---在不同细胞之间存在差异。他推断与人基因组不存在同源性的寡核苷酸可能干扰这种修复过程，从而提高基因敲除成功率。
CRISPR-Cas9基因编辑可以认为是切割和DNA修复之间的竞争：一旦Cas9进行切割，细胞精确地替换受到切割的DNA，随后Cas9再次切割这种受到替换的DNA，从而陷入一种无尽的切割和修复的循环，直到这些修复酶发生错误，这些基因最终都失去功能。这些寡核苷酸可能会降低这种修复过程的保真度，或者说让细胞切换到一种更加容易发生的修复过程，从而允许Cas9更容易让基因发生断裂。本发明可以极大的提高了CRISPR/Cas9敲除效率，同时使该技术更加的简单、易操作。
发明内容
本发明属于分子生物学与生物医学技术领域，本发明涉及一种提高CRIPSR/Cas9靶向敲除基因NHEJ效率的方法。具体来说，本发明设计筛选出高效干扰pten基因表达的小分子干扰RNA，使用CRISPR/Cas9系统靶向敲除基因时，共转染该小分子RNA，可以有效的提高靶基因的NHEJ效率。在细胞多个靶点验证，均有明显提高，该方法成本低、操作简单、效率高。对于CRISPR/Cas9技术的效率和应用具有很好的促进作用。
本发明技术方案如下：
1、高效干扰人pten基因表达的小分子干扰RNA，设计、合成以及筛选；
2、 设计、构建敲除人pten基因的CRIPSR/Cas9载体，并共转染筛选得到的小分子干扰RNA，通过检测靶位点产生的NHEJ效率，确认CRISPR/Cas9载体共转染小分子干扰RNA是否可以提高NHEJ效率；
3、 将小分子干扰RNA应用于多个CRISPR/Cas9靶位点编辑验证，确认小分子干扰RNA提高NHEJ效率的准确性。
附图说明
为了使本发明的目的、技术方案和有益效果更加清楚，本发明提供如下附图：
图1 为反转录PCR检测pten基因的表达量（其中M为DL2000，1为pten-RNAi-1，2为pten-RNAi-1， 3为pten-RNAi-3，4为pten-RNAi-4，5为pten-RNAi-5，PCR扩增264bp大小片段）；
图2为Lin28a两个靶点区域PCR产物T7 Endonuclease I酶切产物，琼脂糖凝胶电泳检测结果（其中A组为小干扰RNA+ PX458-Lin28A-T1 ，B组为小干扰RNA+ PX458-Lin28A-T5，C组为PX458-Lin28A-T1，D组为PX458-Lin28A-T5，E组为小干扰RNA，F组为PBS）。
具体实施方式
下面将结合附图，对本发明的优选实施例进行详细的描述。实施例中未注明具体条件的实验方法，通常按照常规条件，例如分子克隆实验指南（第三版，J. 萨姆布鲁克等著）中所述的条件，或按照制造厂商所建议的条件。
根据NCBI数据库发现基因PTEN的CDS区域，并结合NCBI Primer-BLAST设计引物序列，同时根据专利（201611252179.5）合成lin28a基因靶位点区域扩增引物，序列如下：
通过在线软件设计出5条针对PTEN基因的小干扰RNA序列，并合成，其对应的RNA序列如SEQ ID NO .1 --SEQ ID NO .5所示。
实验具体步骤
转染前3天，复苏人胚胎肾细胞（293T细胞株，中科院上海细胞库），将细胞放入加有10%的FBS+DMEM培养瓶中，于37℃、5% CO2的培养箱中培养，转染前1天，传代培养复苏细胞；
将培养293T细胞T75瓶中的培养基吸净，加入2 mL 4℃冰箱取出的0.25%胰酶，使其均匀覆盖瓶底，置于37℃培养箱中3~5 min，取出，摇晃可发现细胞于底部脱离，将其全部晃下，加入3 mL 37℃水浴中预热的10％FBS+DMEM培养基，用10 mL移液管进行吹打，将细胞悬浮液收集、离心；
转染前1天，接种2×104个293T细胞至48孔培养板中，过夜培养，使转染时的细胞密度达到80％；
实验分组为pten-RNAi-1转染组，pten-RNAi-2转染组，pten-RNAi-3转染组，pten-RNAi-4转染组，pten-RNAi-5转染组，PBS转染组（对照组）每组6个复孔；
小干扰RNA与转染试剂：每孔加入小干扰RNA 20pmol：转染试剂1.0μL：用50µL Opti-MEM稀释，轻轻吹吸3~5 次混匀，室温下静置20 min，随后加入培养皿中进行细胞转染（详细步骤参见Lipofectamine 2000使用说明书），转染操作完成后，经过37℃培养4h后，用10%FBS+DMEM培养基的完全培养基换液，继续培养；
1.将细胞培养至24 h，使用PBS清洗一次细胞，加Beyozol Reagent 裂解细胞，室温放置5 min；
2.每管加入0.2mL氯仿，剧烈震荡离心管15 s，室温放置3 min，离心12000g，4℃ 15min。
3.离心后取上清，加入等体积的异丙醇，冰浴10 min，离心12000g，4℃ 10 min；
4.离心后移去上清，用1mL75%的乙醇洗涤，然后离心7400g，4℃ 5分钟；
5.移去乙醇溶液，自然晾干5分钟，再加入DEPC水溶解RNA；
6.检测RNA质量；
7.将提好的Total RNA作为模板，用Takara PrimeScript II 1st strand cDNASynthesis Kit货号6210A/B合成cDNA；
逆转录操作步骤：
（1）准备混合体系如下：
（2）65℃孵育5min；立刻放置冰上冷却；
（3）准备反应体系如下：
（4）轻轻混匀；
（5）在42℃孵育60 min；
（6）95℃孵育5 min是逆转录酶失活并终止反应。
8.根据目的基因Strn设计1对引物进行PCR，反应体系如下：
9.电泳，取5 μL PCR产物，1.0% agrose regular，100V 1X TAE buffer，20 min；
10.根据电泳结果判断5个小干扰RNA转染处理的细胞中pten基因的表达量，如附图1所示，
从PCR条带亮度可以看出：2、4、5号条带较弱，具有明显的干扰效率。
根据专利（201611252179.5）中筛选到的敲除Lin28a基因CRISPR/Cas9 两个靶位点序列，分别构建出对应的CRIPSR/Cas9载体，根据专利（201611252179.5）中名称为PX458-Lin28A-T1和PX458-Llin28A-T5，构建载体详细步骤参照专利（201611252179.5）。
共转染，转染前1天，接种2×104个293T细胞至48孔培养板中，过夜培养，使转染时的细胞密度达到80％。pten基因小干扰RNA分别与CRISPR/Cas9载体PX458-lin28A-T1和PX458-lin28A-T5共转染人293T细胞株，具体实验分组：A组为小干扰RNA+ PX458-Lin28A-T1 ，B组为小干扰RNA+ PX458-Lin28A-T5，C组为PX458-Lin28A-T1，D组为PX458-Lin28A-T5，E组为小干扰RNA，F组为PBS。
配置RNA与转染试剂：每孔加入小干扰RNA 20 pmol：转染试剂=1.0 μL：用50 µLOpti-MEM稀释，轻轻吹吸3~5 次混匀，室温下静置20 min。
PX458-Lin28A-T1/ PX458-Lin28A-T5：转染试剂=0.8 μg：2μL用50 μLOpti-MEM稀释，轻轻吹吸混匀，室温下静置20 min。
根据实验分组，采用上述配制方法将小干扰RNA与质粒转染293T细胞。
转染操作完成后，经过37℃培养4 h后，用10%FBS+DMEM培养基的完全培养基换液，继续培养。待将细胞培养至24 h生效率。
以提取的DNA为模板，扩增靶点序列，采用T7 Endonuclease I酶切鉴定，验证靶点序列突变情况。
PCR反应体系如下：
PCR扩增程序：95℃预变性3 min；95℃变性30 s，58℃退火30 s，72℃延伸40 s，30个循环后72℃延伸5 min，最后4℃保温。
PCR产物用T7 Endonuclease I 37℃水浴酶切1h，酶切体系如下：
酶切产物经琼脂糖凝胶电泳检测分析，果图2显示各组（A组、 B组、C组、D组、E组和F组）靶位点NHEJ效率，软件分析条带的灰度值 InDel(%)：A组7.8%、B组8.6%、C组为2.3%、D组为1.1%，小干扰RNA对于CRISPR/Cas9靶向敲除Lin28a基因的两个靶位点的形成NHEJ效率分别提高了3.39倍和7.8倍。
SEQUENCE LISTING
<110> 重庆高圣生物医药有限责任公司
<120> 一种提高CRIPSR/Cas9靶向敲除基因产生非同源性末端接合效率的方法
<130> 2017
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Claims (6)
1.本发明涉及一种可以有效提高CRISPR/Cas9技术在敲除基因应用的方法，其特征在于：转染CRISPR/Cas9载体的同时，共转染本发明中涉及的小分子干扰RNA，可以有效提高CRISPR/Cas9系统靶位点形成的NHEJ的效率，具体包括如下步骤：
（1）设计pten基因的gRNA，每个基因3个靶位点，合成对应的oligo和用BbsI酶切过的PX458 载体进行连接，将连接产物转化到大肠杆菌DH5a中，涂布于带有氨苄青霉素抗性的LB平板上，筛选阳性菌落，提取阳性菌落质粒进行分析及测序，确认gRNA表达载体构建正确；
（2）将步骤（1）构建的gRNA载体与小干扰RNA共转染转染293T细胞，另取构建的gRNA载体转染293T细胞（作为对照组）；
（3）将步骤（2）转染24~48小时，细胞经流式分选，筛选出GFP标记的转染阳性细胞；
（4）提取基因组，PCR扩增靶位点区域片段，将PCR产物退火，并用T7E1酶切，采用琼脂糖凝胶电泳检测，并进行灰度分析（indel）检测NHEJ效率，检测结果发现共转染小干扰RNA可以有效提高CRISPR/Cas9在靶位点形成NHEJ效率为3.39~7.8倍。
2.根据权利要求1所述的小分子干扰RNA，其特征在于：该小分子RNA具有有效下调人pten基因的表达功能。
3.根据权利要求1所述的小分子干扰RNA，其对应的RNA序列如序列表SEQ ID NO. 3-4、SEQ ID NO. 7-8和SEQ ID NO. 9-10所示。
4.根据权利要求1所述的小分子干扰RNA，为权利要求3所述的3条小分子RNA序列的等比混合物。
5.本发明涉及的小干扰RNA可以有效提高CRISPR/Cas9系统在293T细胞中靶向敲除人pten基因的NEHJ效率。
6.一种CRISPR/Cas9高效靶向敲除人pten基因的技术方法。
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