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, once validated in cell culture, and analyse them as described above in order to determine the genetic basis of appetite regulation; a major factor in determining susceptibility to obesity.
1. McEwan A, Hay EA, Marini P, Turnbull Y, Wilson D, McIntosh A, et al. CRISPR disruption of a conserved polymorphic enhancer associated with increased alcohol intake in women reveals its functional role in ethanol and fat consumption. American Journal Human Genetics. 2018; Submitted.
2. Modzelewski AJ, Chen S, Willis BJ, Lloyd KCK, Wood JA, He L. Efficient mouse genome engineering by CRISPR-EZ technology. Nat Protoc. 2018;13(6):1253-74.
3. Speliotes EK, Willer CJ, Berndt SI, Monda KL, Thorleifsson G, Jackson AU, et al. Association analyses of 249,796 individuals reveal 18 new loci associated with body mass index. Nat Genet. 2011;42(11):937-48.
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