Leveraging CRISPR RNA-guided DNA Transposases for Gene Insertion at the CFTR Locus

项目摘要 尽管自CRISPR-Cas9出现以来，基因编辑技术取得了许多令人兴奋的进展， 由于无法催化可编程和可预测的大DNA插入， 有效载荷而不产生双链断裂（DSB）。DSB生成编辑工具的使用 发现了大量的安全性问题，包括存在大的靶基因组缺失和脱靶基因组缺失。 插入和删除。此外，基于Cas9的方法用于编辑由大的 基因突变的多样性，如囊性纤维化（CF），需要针对每个等位基因定制的个体指导RNA， 这使得该技术不能作为广泛使用的临床工具。对于CF，优化基因编辑 具有独立于DSB、可编程、有针对性地插入大型货物能力的技术将 允许出现通用CF治愈，而不管患者的突变，通过插入功能性拷贝的 CFTR cDNA在内源基因座。 CRISPR相关转座子的最近发现和发展提供了一个令人兴奋的新的 以高整合效率和几乎没有脱靶事件插入大遗传货物（> 10 kb）的策略。 该建议旨在系统地优化RNA引导的转座酶，称为INTEGRATE，在人类中， 细胞，以实现治疗相关的编辑效率，并将其应用于普遍纠正 CFTR基因突变。目标1将集中于通过改进DNA插入效率的严格优化。 蛋白质递送和INTEGRATE效应复合物的共定位。这一目标的一个关键组成部分是 全面评估目标上和目标外编辑事件。目的2将指导全长的靶向插入 在人支气管上皮细胞内源性基因位点的CFTR cDNA，并定量CFTR的产生。 成熟CFTR和生理CFTR离子通道活性的恢复。这项研究将为 继续开发RNA引导的转座酶基因编辑技术， CF的普遍校正，这对其他遗传疾病具有广泛的适用性。

PROJECT SUMMARY Despite the many exciting advances in gene editing technologies since the advent of CRISPR-Cas9, the field has been hampered by an inability to catalyze programmable and predictable insertion of large DNA payloads without generating double-strand breaks (DSBs). The use of DSB-generating editing tools has unearthed substantial safety concerns, including the presence of large on-target genomic deletions and off-target insertions and deletions. Additionally, Cas9-based approaches for editing diseases that are caused by a large diversity of mutations in a gene, like cystic fibrosis (CF), require individual guide RNAs tailored to each allele, which makes this technology prohibitive as a broadly accessible clinical tool. For CF, optimizing a gene editing technology with the ability to perform DSB-independent, programmable, targeted insertion of large cargos would allow the advent of a universal CF cure regardless of a patient’s mutation(s) by inserting a functional copy of CFTR cDNA at the endogenous locus. The recent discovery and development of CRISPR-associated transposons offers an exciting new strategy to insert large genetic cargos (>10kb) with high integration efficiencies and virtually no off-target events. This proposal aims to systematically optimize RNA-guided transposases, referred to as INTEGRATE, in human cells to achieve therapeutically relevant editing efficiencies, and to apply them for the universal correction of CFTR gene mutations. Aim 1 will focus on a rigorous optimization of DNA insertion efficiencies by improving protein delivery and colocalization of the INTEGRATE effector complex. A critical component of this aim will be comprehensively assessing on- and off-target editing events. Aim 2 will direct the targeted insertion of full-length CFTR cDNA at the endogenous gene locus in human bronchial epithelial cells, and quantify the production of mature CFTR and the restoration of physiologic CFTR ion channel activity. This study will pave the way for the continued development of RNA-guided transposase gene editing technologies for the DSB-independent universal correction of CF, which carries broad applicability to other genetic diseases.