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

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.