Date of Award
© 2021 Noah C. Cheng. This work is freely available courtesy of the author. It may be used under the terms of the Creative Commons Attribution-NoDerivatives 4.0 International (CC BY-ND 4.0) license. For all other uses, please contact the copyright holder.
Creative Commons License
This work is licensed under a Creative Commons Attribution-No Derivative Works 4.0 International License.
Bachelor of Arts
Amy Cheng Vollmer
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:  nucleofection of HDR repair templates into cells and subsequent treatment with VLPs carrying Cas9 RNPs (Cas9 VLPs),  treatment with Cas9 VLPs loaded with repair templates through electroporation,  treatment with VLPs packaging both Cas9 RNPs and a lentiviral genome that encoded the repair template, and  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.
Cheng, Noah C. , '21, "Virus-Like Particles Enable Low Levels of CRISPR-Cas9-Mediated Homology-Directed Repair" (2021). Senior Theses, Projects, and Awards. 165.