CRISPR-based epigenetic modifier

 描述(申请人提供)：这个项目的目标是设计一个易于使用、高度特异和有针对性的修饰因子工具箱，可以以可预测的方式操纵表观基因组。肿瘤抑制基因的启动子是高甲基化的，在癌细胞中不活跃。表观基因组有利于在药物存在的情况下存活，或癌基因的异位表达，使化疗无效，肿瘤细胞生长。更好地了解癌症表观遗传学对于癌症研究和治疗很重要。然而，我们对这些修改的功能后果的理解仍然远远不完整，我们以有针对性的方式设计具体修改的能力还处于初级阶段。影响表观遗传信息的药物已用于癌症的临床治疗，但由于缺乏特异性，导致基因表达的广泛变化。在将表观遗传酶连接到可编程DNA结合域(如锌指(ZF)或TALE)方面取得了有限的成功。由于克隆引导RNA的简便性和高度的结合特异性，人们对使用更有用的CRISPR/Cas9系统来修饰表观基因组很感兴趣。然而，到目前为止报道的大约20个表观遗传修饰物中，只有两个使用了CRISPR/Cas9系统。这不是因为缺乏兴趣或努力；它更可能反映了在使用CRISPR/CAS9系统时遇到的一个未解决的问题。我们假设，针对DNA的dCas9的重要结构特征不同于ZF或TALE，并且需要改变过去的设计策略来创建CRISPR表观遗传修饰物。我们认为这是一个需要解决的重要问题，以便CRISPR/CAS9系统可以用来以特定部位的方式改变表观基因组。该项目的目标1是设计能够改变特定表观遗传标记的基于CRISPR/CAS9的系统。我们假设，直接修饰DNA的表观遗传编写器和擦除器被与DNA结合的dCas9的结构特征所抑制。为了绕过这个问题，我们将使用替代的设计策略将表观遗传修饰复合体系在dCas9上，并测量它们改变特定标记的能力。在目标2中，我们将描述CRISPR/Cas9表观遗传工具如何改变基因表达。我们假设，更长的表观遗传修饰区域应该会导致监管的逐步增加和更大的持久性。我们将评估实现强大的基因调控所需的修饰长度，确定这些因素是否提供分级或阈值响应，并检查新的表观遗传和基因表达模式持续多长时间。最后，我们将检查全基因组范围内表观遗传标记和基因表达的变化。这项提案中开发的工具和信息将形成一个基础，在此基础上可以开发许多在肿瘤发生、耐药性和新的癌症治疗方面的应用。

 DESCRIPTION (provided by applicant): The aim of this project is to engineer a toolbox of easy-to-use, highly specific and targetable modifying factors that can manipulate the epigenome in a predictable manner. The promoters of tumor suppressor genes are hypermethylated and inactive in cancer cells. Epigenomes favoring survival in the presence of a drug, or ectopic expression of an oncogene, render chemotherapeutics ineffective and tumor cells to grow. A better understanding of cancer epigenetics is important for cancer research and therapy. However, our understanding of the functional consequences of these modifications is still far from complete and our ability to engineer specific modifications in a targeted manner is in its infancy. Drugs affecting epigenetic information are in clinical use for cancer, but cause broad changes in gene expression due to lack of specificity. Limited success has been achieved in attaching epigenetic enzymes to programmable DNA-binding domain such as zinc fingers (ZFs) or TALEs. Because of the ease in cloning guide RNAs and due to a high binding specificity, there is much interest in using the far more useful CRISPR/Cas9 system to modify the epigenome. However, of the ~20 epigenetic modifiers reported to date only two have used the CRISPR/Cas9 system. This has not been due to lack of interest or effort; it more likely reflects an unresolved problem encountered when employing the CRISPR/Cas9 system. We hypothesize that important structural features of dCas9 targeted to DNA are different than for ZFs or TALEs, and that a change in past design strategies is required to create CRISPR epigenetic modifiers. We feel that this is an important problem that needs to be solved so that the CRISPR/Cas9 system can be used to alter the epigenome in a site-specific manner. Aim 1 of this project is to design CRISPR/Cas9-based systems that can alter specific epigenetic marks. We hypothesize that epigenetic writers and erasers that directly modify DNA are inhibited by structural features of dCas9 bound to DNA. To circumvent this problem, we will use alternative design strategies to tether epigenetic modifying complexes to dCas9, and measure their ability to alter specific marks. In Aim 2, we will characterize how CRISPR/Cas9 epigenetic tools alter gene expression. We hypothesize that longer regions of epigenetic modification should lead to a graded increase in regulation, and greater persistence. We will assess the length of modification needed to achieve robust gene regulation, determine if the factors provide a graded or threshold response, and examine how long the new epigenetic and gene expression patterns persist. Finally, we will examine genome-wide changes in epigenetic marks and gene expression. The tools and information developed in this proposal will form a foundation from which numerous applications in tumorigenesis, drug resistance, and new cancer therapeutics could be developed.