Improving CRISPR Based Prime Gene Editing For Large Fragment Edits

项目概述：基因组编辑工具提供了研究和操纵基因组的方法。这 允许开发细胞和动物模型来研究许多疾病的病理基础， 疾病在过去十年中，基于CRISPR-Cas的基因组编辑工具的快速发展推动了 在生物科学的进步，并已显示出令人难以置信的效用，在广泛的领域，从基本的 研究到临床试验。Prime Editing（PE）是一种新开发的基于CRISPR-Cas9的工具， DNA序列，并已被证明可以执行精确的靶向插入，删除，以及所有12种可能的 不需要DSB的点突变类型。虽然这项技术有很大的希望，但进一步的研究 需要了解DNA修复机制，从而为基因组的编辑和传递策略提供有效的指导。 PE系统允许该技术的广泛适用性。 在我的初步研究中，我已经表明，PE系统的关键组件的调制提高了编辑 效率。此外，我还将已发表的慢病毒系统浓缩为双重选择慢病毒系统 它适用于多种细胞类型。本研究的目标是进一步开发一个体育平台 能够对功能序列插入进行大片段编辑。在目标1中，我将使用结构引导 在一个实施方案中，可以通过诱变来产生能够更好地适应大片段编辑的PE突变体。在aim 2中，我将使用我的 生成的工具，用功能序列内源性标记BRD 4同种型，以表征它们在 白血病尽管BRD 4是临床试验中具有超过20种抑制剂的治疗靶点，但这些抑制剂在临床试验中仍然存在。 非特异性药物靶向BET家族的几个成员和所有BRD 4亚型，这是正常代谢所需的。 细胞功能由于该靶标缺乏市售的同种型特异性ChIP级抗体， 独特的靶向C末端区域，它作为一个实用的和疾病相关的目标，以证明 的概念。最终，这种方法的开发将为CRISPR-Cas9工具箱提供补充 能够完成许多其他任务。

Project Summary: Genome editing tools provide methods to study and manipulate the genome. This has allowed for the development of cellular and animal-based models to study the pathological basis of many diseases. Over the last ten years, the rapid development of CRISPR-Cas based genome editing tools has driven advances in the biological sciences and has shown incredible utility in a wide range of fields ranging from basic research to clinical trials. Prime Editing (PE) is a newly developed CRISPR-Cas9 based tool that directly rewrites DNA sequences and has been shown to perform precise targeted insertions, deletions, and all 12 possible classes of point mutations without requiring DSBs. While this technology holds great promise, further study is needed to understand DNA repair mechanisms that result in productive editing and delivery strategies for the PE system to allow for broad applicability of this technology. In my preliminary studies, I have shown that modulation of key components of the PE system improves editing efficiencies. Additionally, I have condensed the published lentiviral system to a dual selection lentiviral system that is suitable for a wide range of cell types. The goal of this study is to further develop a PE platform capable of large fragment edits for functional sequence insertions. In aim 1, I will use structure-guided mutagenesis to generate a PE mutant better able to accommodate large fragment edits. In aim 2, I will use my generated tool to endogenously tag BRD4 isoforms with a functional sequence to characterize their roles in leukemia. Even though BRD4 is a therapeutic target with more than 20 inhibitors in clinical trials, these nonspecific drugs target several members of the BET family and all BRD4 isoforms, which are required for normal cellular function. Since this target lacks commercially available, isoform specific ChIP-grade antibodies and has unique targetable C terminal regions, it serves as a practical and disease relevant target to demonstrate proof of concept. Ultimately, development of this method will provide an addition to the CRISPR-Cas9 toolbox capable of numerous other tasks.