In vivo enhancer manipulation using CRISPR genome editing

使用 CRISPR 基因组编辑进行体内增强子操作

• 批准号: 2282111
• 金额: --
• 依托单位: University of Aberdeen
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2282111
• 关键词: vivo; enhancer; manipulation; using; CRISPR

Enhancers are enigmatic regions of the genome that preserve health by ensuring the correct expression of genes in the correct tissues. We have shown that disrupting enhancers using CRISPR/CAS9 genome editing is straightforward in mice and produces significant changes in aspects of behaviour such as fat and alcohol intake(1). However, the next challenge is to manipulate specific base pairs within these enhancers in order to determine how they work and to reproduce the effects of human polymorphic variation on their activity. This requires the optimisation of a "knock-in" strategy whereby CAS9 mediated cuts in the genome are repaired in the presence of a repair template that fools mouse embryos into incorporating a new region of DNA into the CAS9 cut site. However, because this homologous repair process is x10 less efficient than the usual non-homologous repair mechanisms used by the embryo we must greatly increase the efficiency of transgenesis that is currently only 10-20%. Recently, a new method involving the introduction of CAS9/CRISPR components using electroporation of embryos, has shown a transgenic rate of almost 100% which considerably increases the chances of producing "knock-in" mouse models (2). This method is also more humane as it uses less mice.We will initially use this technology to humanise the obesity associated polymorphism rs10767664 (p=4.69x10-26) that lies within a highly conserved enhancer called BE5.1 next to the BDNF gene (3). Using embryo microinjection we have already validated the use of specific guide RNAs (gRNA) by producing heterozygous mouse BE5.1 knockout lines. We have also designed and manufactured a single strand repair template that would replace the mouse BE5.1 sequence with the obesity associated human BE5.1 allele in the mouse.In order to humanise BE5.1 mouse embryos will be mixed with a solution containing guideRNA, CAS9 protein and single strand repair template DNA. After electroporation embryo DNA will be analysed using PCR. If we can show that our CRISPR "knock-in" strategy for humanising enhancers via electroporation is effective in-vitro we will repeat the electroporation procedure and transfer embryos to the oviducts of CD1 female mice to produce mouse lines. Once it is confirmed by PCR that correct targeting has occurred, these new mouse lines will be analysed to assess the effects of BE5.1 humanisation on BDNF mRNA expression. Feeding behaviour, weight gain and metabolism will also be analysed in these mice in collaboration with Prof Mirela Delibegovic; an expert in metabolism and diabetes at the University of Aberdeen.Once we have optimised our strategy we will work in collaboration with Prof Andrew McIntosh and Dr Toni-Kim Clarke of the University of Edinburgh who are both geneticists focussed on using large population-based cohorts to find polymorphisms within the human genome that modulate appetite and addictive behaviours. We will use our validated technology to reproduce these polymorphisms in mice and analyse them as described above in order to determine the genetic basis of appetite regulation; a major factor in determining susceptibility to obesity

增强子是基因组中神秘的区域，通过确保正确组织中基因的正确表达来保持健康。我们已经证明，在小鼠中使用CRISPR/CAS9基因组编辑破坏增强子是直接的，并且在脂肪和酒精摄入等行为方面产生重大变化(1)。然而，下一个挑战是操纵这些增强子中的特定碱基对，以确定它们是如何工作的，并重现人类多态性变异对其活性的影响。这需要优化“敲入”策略，即在存在修复模板的情况下修复CAS9介导的基因组切割，该修复模板会欺骗小鼠胚胎将新的DNA区域整合到CAS9切割位点中。然而，由于这种同源修复过程的效率比胚胎通常使用的非同源修复机制低10倍，我们必须大大提高转基因的效率，目前只有10-20%。最近，一种利用胚胎电穿孔引入CAS9/CRISPR组件的新方法显示出几乎100%的转基因率，这大大增加了产生“敲入”小鼠模型的机会(2)。这种方法也更人性化，因为它使用的老鼠更少。我们将首先使用该技术对肥胖相关多态性rs10767664 （p=4.69x10-26）进行人源化，该多态性位于BDNF基因旁边的高度保守增强子BE5.1中(3)。通过胚胎显微注射，我们已经通过产生杂合小鼠BE5.1基因敲除系验证了特异性引导rna （gRNA）的使用。我们还设计并制造了一个单链修复模板，将小鼠BE5.1序列替换为小鼠肥胖相关的人类BE5.1等位基因。为了使BE5.1人源化，将小鼠胚胎与含有引导rna、CAS9蛋白和单链修复模板DNA的溶液混合。电穿孔后用PCR分析胚胎DNA。如果我们能够证明我们的CRISPR“敲入”策略通过电穿孔使人类增强子在体外是有效的，我们将重复电穿孔程序并将胚胎移植到CD1雌性小鼠的输卵管中以产生小鼠品系。一旦通过PCR确认了正确的靶向发生，这些新的小鼠品系将被分析以评估BE5.1人源化对BDNF mRNA表达的影响。与Mirela Delibegovic教授合作，还将分析这些小鼠的摄食行为、体重增加和新陈代谢；阿伯丁大学代谢和糖尿病专家。一旦我们优化了我们的策略，我们将与爱丁堡大学的安德鲁·麦金托什教授和托尼·金·克拉克博士合作，他们都是遗传学家，专注于利用基于大量人群的队列来发现人类基因组中调节食欲和成瘾行为的多态性。我们将使用经过验证的技术在小鼠中复制这些多态性，并如上所述进行分析，以确定食欲调节的遗传基础；这是决定肥胖易感性的主要因素