生成CRISPR / Cas9介导的单等位基因缺失来研究小鼠胚胎干细胞功能增强
Summary
Experimental validation of enhancer activity is best approached by loss-of-function analysis. Presented here is an efficient protocol that uses CRISPR/Cas9 mediated deletion to study allele-specific regulation of gene transcription in F1 ES cells which contain a hybrid genome (Mus musculus129 x Mus castaneus).
Abstract
Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often located distally from the regulated gene in intergenic regions or even within the body of another gene. The position independent nature of enhancer activity makes it difficult to match enhancers with the genes they regulate. Deletion of an enhancer region provides direct evidence for enhancer activity and is the gold standard to reveal an enhancer’s role in endogenous gene transcription. Conventional homologous recombination based deletion methods have been surpassed by recent advances in genome editing technology which enable rapid and precisely located changes to the genomes of numerous model organisms. CRISPR/Cas9 mediated genome editing can be used to manipulate the genome in many cell types and organisms rapidly and cost effectively, due to the ease with which Cas9 can be targeted to the genome by a guide RNA from a bespoke expression plasmid. Homozygous deletion of essential gene regulatory elements might lead to lethality or alter cellular phenotype whereas monoallelic deletion of transcriptional enhancers allows for the study of cis-regulation of gene expression without this confounding issue. Presented here is a protocol for CRISPR/Cas9 mediated deletion in F1 mouse embryonic stem (ES) cells (Mus musculus129 x Mus castaneus). Monoallelic deletion, screening and expression analysis is facilitated by single nucleotide polymorphisms (SNP) between the two alleles which occur on average every 125 bp in these cells.
Introduction
转录调控元件由于异常的基因表达2是发展1和修改这些元素的过程中的基因表达的时空微调可导致疾病的关键。通过全基因组关联研究发现了许多疾病相关的区域是在非编码区，并有转录增强子3-4的功能。识别增强剂并将它们与它们调节是复杂的，因为它们通常位于从它们调节基因几千个碱基远并且可以以组织特异性方式5-6被激活的基因匹配。增强剂的预测通常是基于组蛋白修饰的标记，介体黏着复合物和细胞类型特异性转录结合因子7-10。预测增强剂的验证是最经常通过一个基于矢量的测定，其中所述增强剂激活报告基因11-12的表达进行。这些数据提供了v有关推测的增强子序列的调节潜力aluable信息，但不透露自己的功能其内生基因组范围内或识别它们调节的基因。基因组编辑充当一个强有力的工具来研究由失功能分析在它们的内源上下文转录调控元件的功能。
在基因组编辑，即CRISPR / Cas9基因组编辑系统的最新进展，有利于基因功能的研究。的CRISPR / Cas9系统易于使用和适应性对于许多生物系统。所述Cas9蛋白靶向于由导的RNA（gRNA）13中的基因组中的特定位点。所述SpCas9 / gRNA复杂扫描对其靶基因组序列的基因组中它必须是5'到protospacer相邻基序（PAM）的序列，NGG 14-15。的gRNA到其目标，一个20个核苷酸（nt）的序列与gRNA互补的碱基配对，激活导致域金字塔之戒SpCas9核酸酶活Ë链断裂（DSB）3碱基的序列PAM的上游。特异性是通过在gRNA种子区域完全碱基配对来实现，所述6-12 nt下邻近于PAM;相反地，不匹配5'种子的通常耐受16-17。引入的DSB可以修复或者由非同源末端连接（NHEJ）的DNA修复或同源性定向修复（HDR）mechanisms.NHEJ DNA修复往往造成在目标部位的几个碱基对，可以破坏的插入/缺失（插入缺失）的基因的开放阅读框（ORF）。以产生在基因组2 gRNAs，侧翼感兴趣的区域大的缺失，可以使用18-19。这种方法是对聚成基因座控制区或超增强剂它比常规增强剂9,18,20-22较大转录增强子的研究中特别有用的。
单等位基因缺失是研究转录顺 -regulation一个有价值的模型。观察到昌E在转录水平的增强子的单等位基因缺失之后关联到在基因调控该增强剂的不当两个等位基因的转录可能受影响的影响蜂窝健身时可能出现的混杂影响的作用。评估减少的表达是困难的但不区分野生型等位基因的删除的能力。此外，基因分型在每个等位基因缺失而不区分两个等位基因的能力是具有挑战性的，尤其是对大缺失> 10kb的至1兆23，其中它是难以通过PCR扩增整个野生型区域。使用通过杂交小家鼠 129与小家鼠castaneus生成的F1 ES细胞的允许两个等位基因通过等位基因特异性PCR 18,24区别开来。在这些细胞中的基因组杂交便于等位基因特异缺失筛选和表达分析。上平均有一个SNP位这两个基因组之间的每一个125 bp的为表达和基因分型提供在引物设计的灵活性的分析。一种SNP的存在可以影响引物的熔化温度（T M）与靶实时定量PCR（qPCR的）扩增特异性允许两个等位基因25的歧视。此外，引物的3'末端中的一个错配极大地影响DNA聚合酶从引物防止不期望的等位基因靶26的扩增延伸的能力。描述在下面的协议是使用CRISPR / Cas9基因组编辑系统（ 图1）大于1 kb的等位基因特异性增强缺失和随后的表达分析使用F1 ES细胞。
图1.增强删除使用CRISPR / Cas9研究 顺 -reg基因表达的ulation。（A）中由小家鼠 129和小家鼠castaneus之间的交叉产生的F1 ES细胞用于允许等位基因特异性缺失。 （B）中的两个导向的RNA（gRNA）用于诱导增强子区的一个大Cas9介导的缺失。 （C）的引物组被用于识别大的单-和双等位基因缺失。橙色引物是内引物，紫色引物外侧的引物和绿色的引物的gRNA侧翼引物。 （D）基因表达的变化是使用等位基因特异性qPCR的监控。俄罗斯足协表示相对荧光单位。 请点击此处查看该图的放大版本。
Protocol
Representative Results
Discussion
CRISPR / Cas9介导的基因组编辑技术为基因改造一个简单，快捷，廉价的方法。这里详细生成和分析单等位基因缺失的增强功能性增强特性的方法发生在F1小鼠细胞单核苷酸多态性的优势的。这种类型的方法的优点是：1）单等位基因增强剂缺失不产生时的临界增强剂是从两个等位基因缺失， 即 ，在规定的基因导致细胞杀伤力的蛋白水平大大降低的或改变的发生的混杂影响表型; 2）如果单等位…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
We would like to thank all the members of the Mitchell lab for helpful discussions. This work was supported by the Canadian Institutes of Health Research, the Canada Foundation for Innovation and the Ontario Ministry of Research and Innovation (operating and infrastructure grants held by JAM).
Materials
| Phusion High-Fidelity DNA Polymerase | NEB | M0530S | high fidelity DNA polymerase used in gRNA assembly |
| Gibson Assembly Master Mix | NEB | E2611L | |
| gRNA_Cloning Vector | Addgene | 41824 | A target sequence is cloned into this vector to create the gRNA plasmid |
| pCas9_GFP | Addgene | 44719 | Codon-optimized SpCas9 and EGFP co-expression plasmid |
| AflII | NEB | R0520S | |
| EcoRI | NEB | R3101S | |
| Neon Transfection System 100 µL Kit | Life Technologies | MPK10096 | Microporator transfection technology |
| prepGEM | ZyGEM | PT10500 | genomic DNA extraction reagent |
| Nucleo Spin Gel & PCR Clean-up | Macherey-Nagel | 740609.5 | |
| High-Speed Plasmid Mini Kit | Geneaid | PD300 | |
| Maxi Plasmid Kit Endotoxin Free | Geneaid | PME25 | |
| SYBR select mix for CFX | Life Technologies | 4472942 | qPCR reagent |
| iScript cDNA synthesis kit | Bio-rad | 170-8891 | Reverse transcription reagent |
| 0.25% Trypsin with EDTA | Life Technologies | 25200072 | |
| PBS without Ca/Mg2+ | Sigma | D8537 | |
| 0.5M EDTA | Bioshop | EDT111.500 | |
| HBSS | Life Technologies | 14175095 | |
| 1M HEPES | Life Technologies | 13630080 | |
| BSA fraction V (7.5%) | Life Technologies | 15260037 | |
| Max Efficiency DH5α competent cells | Invitrogen | 18258012 | |
| FBS | ES cell qualified | FBS is subjected to a prior testing in mouse ES cells for pluripotency | |
| DMSO | Sigma | D2650 | |
| Glutamax | Invitrogen | 35050 | |
| DMEM | Life Technologies | 11960069 | |
| Pencillin/Streptomycin | Invitrogen | 15140 | |
| Sodium pyruvate | Invitrogen | 11360 | |
| Non-essential aminoacid | Invitrogen | 11140 | |
| β-mercaptoethanol | Sigma | M7522 | |
| 96-well plate | Sarstedt | 83.3924 | |
| Sealing tape | Sarstedt | 95.1994 | |
| CoolCell LX | Biocision | BCS-405 | alcohol-free cell freezing container |
| CHIR99021 | Biovision | 1748-5 | Inhibitor for F1 ES cell culture |
| PD0325901 | Invivogen | inh-pd32 | Inhibitor for F1 ES cell culture |
| LIF | Chemicon | ESG1107 | Inhibitor for F1 ES cell culture |
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