Virus-Like Particles Enable Low Levels of CRISPR-Cas9-Mediated Homology-Directed Repair
Year: 2021
Type: dissertation
Abstract: The development of efficient and safe delivery platforms for CRISPR-Cas9 genome editing tools is one of the largest challenges for in vivo therapeutic genome editing. Virus-like particles (VLPs), which harness viral packaging and delivery mechanisms, have been shown to efficiently deliver CRISPR-Cas9 ribonucleoprotein complexes (RNPs) to cells. Here, we sought to elucidate whether VLPs can deliver the necessary components for CRISPR-Cas9-mediated homology-directed repair (HDR). We tested four different methods to induce HDR in a reporter cell line using VLPs: [1] nucleofection of HDR repair templates into cells and subsequent treatment with VLPs carrying Cas9 RNPs (Cas9 VLPs), [2] treatment with Cas9 VLPs loaded with repair templates through electroporation, [3] treatment with VLPs packaging both Cas9 RNPs and a lentiviral genome that encoded the repair template, and [4] treatment with Cas9 VLPs and VLPs carrying just the HDR template encoded on the lentiviral genome (dual-VLP system). We found that VLPs enabled CRISPR-Cas9-mediated HDR, albeit at low frequencies. HDR efficiency was highest when the HDR template was nucleofected into the cells, but appreciable levels of HDR was also observed using the dual-VLP system. This work suggests that VLPs are a viable delivery platform for the necessary components for CRISPR-Cas9- mediated HDR, but considerable improvements in the platform must be made before it is used in therapeutic settings.
Author: Noah Cheng
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W3184329283