A Baculoviral ‘Swag Bag’ Protein and DNA Delivery Toolkit for CRISPR-based Editing of Human Genomes

Abstract

Recent advances in genome engineering technologies notably driven by the discovery of clustered regularly interspaced short palindromic repeats and associated protein 9 (CRISPR- Cas9) provide highly promising avenues for treating a broad range of monogenic and complex inherited diseases. The delivery of CRISPR components to target cells is key to ensure both efficiency and specificity of the genomic intervention. Precision genome editing typically requires simultaneous delivery of Cas9, single guide RNA (sgRNA) and a DNA donor that is used as a repair template for integration. Prolonged exposure to the Cas9 nuclease favours off- target editing, which can result in detrimental, even catastrophic outcomes. This can be overcome by transient intervention by means of Cas9 ribonucleoproteins (RNPs) delivery, which efficiently reduces such undesired side effects. However, the lack of delivery systems compatible with RNP delivery, large DNA payloads co-delivery as well as large scale manufacturing are currently limiting the progress of CRISPR-based therapies to the clinic. In this thesis, a new genome editing delivery system, named Baculoviral Swag Bag (BSB) was created and engineered to cope with the unique requirements of next-generation genome editing technologies. BSBs are baculovirus viral particles, in which Cas9 protein or RNPs are entrapped during their production process in insect cells. With this new delivery system, rapid and efficient genome editing in cultured cells is accomplished, significantly outperforming plasmid DNA transfection techniques. The large DNA cargo capacity of the baculovirus was exploited in a proof-of-concept for targeted 2 kb insertion using “all-in-one” BSBs as a safe and promising tool for complex genome engineering applications. Furthermore, the versatility of this new BSB system was demonstrated by delivering a complete cytidine base editor system (BE3), inducing high levels of base conversion without DNA double-stranded breaks. Finally, a chemically inducible heterodimerization system was implemented, considerably improving protein packaging into BSBs, further expanding the scope of applications of this delivery system. In summary, BSBs provide a safe, versatile and efficient alternative to current delivery systems for future CRISPR-based genome editing therapies.

Date of Award	2 Dec 2021
Original language	English
Awarding Institution	The University of Bristol
Supervisor	Imre Berger (Supervisor) & Mark S Dillingham (Supervisor)