Application of CRISPR technologies for the efficient development of models of human disease

CRISPR/Cas9是一种可编程的位点特异性核酸酶，可用于以精确和有效的方式操纵DNA序列。利用这些“基因组编辑”方法，基因可以从基因组中剔除，以便在干细胞和动物模型中评估它们的功能。此外，已发现与特定疾病进展或易感性相关的单核苷酸变异也可以引入这些系统，从而允许在实验室内对人类疾病模型进行研究。CRISPR/Cas9系统不仅是一种有效的基因组编辑工具，如果催化失活，它还可以作为一种非常有效的可编程位点特异性DNA结合蛋白。使用该系统，可以上调或下调基因表达的效应域可以被招募到基因启动子和调控区域，从而允许对基因表达水平进行可调的操纵-即所谓的CRISPRa（激活）和CRISPRi（失活）技术。由于越来越清楚的是，影响疾病的大部分基因组变异位于基因组的调控区域内，因此开发用于操纵模型系统中基因表达的强大系统将使人们能够深入了解基因表达网络中的扰动如何成为疾病易感性和进展变化的基础。该研究项目将重点研究实施和结合CRISPR系统的有效方法，用于靶向缺失、抑制和激活，以模拟疾病相关的基因组变化。在干细胞和小鼠胚胎中工作，该项目旨在探索人类疾病精确模型的新可能性，特别侧重于降低疾病模型生成的动物成本。

CRISPR/Cas9 is a programmable site-specific nuclease that can be used to manipulate the sequence of DNA in a precise and efficient manner. Using these "genome editing" approaches, genes can be knocked out of the genome to allow an assessment of their function in both stem cell and animal models. Furthermore, single nucleotide variants which have been found to associate with a particular disease progression or susceptibility can also be introduced into these systems, allowing models of human disease to be interrogated within the laboratory.As well as being an effective genome-editing tool, the CRISPR/Cas9 system, if rendered catalytically inactive, serves as a very effective programmable site-specific DNA binding protein. Using this system, effector domains which can either up- or down-regulate gene expression can be recruited to gene promoters and regulatory regions, allowing tuneable manipulation of gene expression levels - so called CRISPRa (activation) and CRISPRi (inactivation) technology. Since it is becoming clear that much of the genomic variation which influences disease, lies within regulatory regions of the genome, the development of robust systems for manipulating gene expression in models systems will enable insights into how perturbations in gene expression networks underlie changes in disease susceptibility and progression.This research project will focus on efficient methods of implementing and combining CRISPR systems for targeted deletion, inhibition and activation to model disease-associated genomic changes. Working in both stem cells and mouse embryos, the project aims to explore new possibilities for accurate models of human disease, with a particular focus on reducing the animal cost of disease model generation.