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

项目总结 尽管自CRISPR-Cas9问世以来，基因编辑技术取得了许多令人兴奋的进步，但 菲尔德一直受到无法催化可编程和可预测的大DNA插入的阻碍 不产生双链断裂(DSB)的有效载荷。DSB生成编辑工具的使用有 发现了大量的安全问题，包括存在大量靶上基因组缺失和靶外缺失 插入和删除。此外，基于Cas9的方法用于编辑由大型 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量身定做的单独引导RNA， 这使得这项技术作为一种广泛可用的临床工具令人望而却步。对于CF，优化一个基因编辑 能够执行独立于DSB的、可编程的、有针对性的大型货物插入的技术将 允许出现通用的CF治愈方法，而不考虑患者的突变(S)，方法是插入 Cftr基因位于内源基因座。 最近CRISPR相关转座子的发现和发展提供了一个令人兴奋的新的 策略插入大的遗传货物(&gt；10kb)，整合效率高，几乎没有偏离目标的事件。 这项建议旨在系统地优化RNA引导的转座酶，称为整合，在人类 细胞以实现与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将专注于通过提高DNA插入效率的严格优化 整合效应器复合体的蛋白质传递和共定位。这一目标的一个关键组成部分将是 全面评估目标上和目标外的编辑事件。目标2将定向插入全长 人支气管上皮细胞内源性基因的cDNAc DNA，并定量测定其产物 成熟的cftr和生理cftr离子通道活性的恢复。这项研究将为 RNA引导的转座酶基因编辑技术在DSB非依赖型中的持续发展 对CF的普遍纠正，对其他遗传性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.