In vivo enhancer manipulation using CRISPR genome editing

增强子是基因组中的神秘区域，通过确保基因在正确的组织中正确表达来保持健康。我们已经证明，在小鼠中使用CRISPR/Cas9基因组编辑扰乱增强子是直接的，并在脂肪和酒精摄入量等行为方面产生显著变化(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，并重现人类多态变异对其活性的影响。这需要优化“敲入”策略，即在修复模板存在的情况下，修复Cas9介导的基因组切割，该修复模板可以愚弄小鼠胚胎，使其将新的DNA区域整合到Cas9切割位置。然而，由于这种同源修复过程比胚胎通常使用的非同源修复机制的效率低10倍，我们必须大大提高转基因效率，目前转基因效率仅为10-20%。最近，一种通过胚胎电穿孔引入Cas9/CRISPR组件的新方法显示出几乎100%的转基因比率，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。我们最初将使用这项技术来人性化肥胖相关的多态rs10767664(p=4.69x10-26)，该多态位于BDNF基因(3)旁边的高度保守的增强子BE5.1中。利用胚胎显微注射，我们已经通过产生杂合的小鼠BE5.1基因敲除系来验证特定引导RNA(GRNA)的使用。我们还设计和制造了一种单链修复模板，可以用肥胖相关的人BE5.1等位基因在小鼠体内取代小鼠的BE5.1序列。为了使BE5.1小鼠胚胎人性化，我们将把BE5.1小鼠胚胎与含有guideRNA、Cas9蛋白和单链修复模板DNA的溶液混合。电穿孔后的胚胎DNA将用聚合酶链式反应进行分析。如果我们能证明我们的CRISPR通过电穿孔人源化增强剂的“敲入”策略在体外是有效的，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，以产生小鼠系。一旦通过聚合酶链式反应确认已经发生了正确的靶向，将对这些新的小鼠品系进行分析，以评估BE5.1人源化对BDNF mRNA表达的影响。阿伯丁大学的新陈代谢和糖尿病专家Mirela Delibegovic教授还将分析这些小鼠的进食行为、体重增加和新陈代谢。一旦我们优化了策略，我们将与爱丁堡大学的安德鲁·麦金托什教授和Toni-Kim Clarke博士合作，他们都是遗传学家，专注于利用大量基于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。我们将使用我们经过验证的技术在小鼠身上复制这些多态，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。

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