Pierre Billon

Cumming School of Medicine, Department of Biochemistry & Molecular Biology, University of Calgary, Canada

From improved Detection to Delivery: Molecular Tools for Efficient CRISPR-based Genome Editing

CRISPR-based genome editing technologies are rapidly advancing basic research and clinical applications, with remarkable recent success in curing genetic diseases and cancer. The advent of new tools, like base editing and prime editing, has expanded the scope and precision of genetic modifications, from single nucleotide changes to gene-sized integrations. However, the targeted delivery of these technologies to specific cells and the accurate detection of desired genetic changes remains critical obstacles for their widespread adoption. In this presentation, I will highlight our recent unpublished work in addressing these challenges.

In particular, we have devised a new method that uses enveloped delivery vehicles to effectively deliver genome editing proteins into mammalian cells. We show that the synergistic combination of the high infectivity mediated by viral-like particles and the robustness editing capabilities of ribonucleoproteins leads to enhanced genome editing efficiencies. We show that the tropism of these vehicles can be tailored to target distinct cell types, achieving high editing efficiencies in hard-to-transfect cells.
Additionally, we have designed a rapid and efficient method for capturing mutations of interest. This method only requires a PCR product to be incubated in an all-in-one reaction for 10 minutes at room temperature. We have devised multiple detection modalities for quantitative, qualitative, and visual detection, and established a rapid clinical diagnostic platform for sickle cell disease that has demonstrated 100% accuracy in identifying pathogenic variants from patients' blood spots and saliva samples. This cost-effective method enriches the toolkit of detection methods for CRISPR-based precision genome editing and clinical applications.

The development of user-friendly molecular tools will ultimately promote the broader accessibility and democratization of genome editing technologies, expediting the realization of their transformative potential.

Selected references

• CRISPR-based genome editing through the lens of DNA repair. Nambiar TS, Baudrier L, Billon P*, Ciccia A*. Mol Cell 2022
• Detection of Marker-Free Precision Genome Editing and Genetic Variation through the Capture of Genomic Signatures. Billon P*, Nambiar TS, Hayward SB, Zafra MP, Schatoff EM, Oshima K, Dunbar A, Breinig M, Park YC, Ryu HS, Tschaharganeh DF, Levine RL, Baer R, Ferrando A, Dow LE, Ciccia A*. Cell Rep 2020
• Stimulation of CRISPR-mediated homology-directed repair by an engineered RAD18 variant. Nambiar TS, Billon P, Diedenhofen G, Hayward SB, Taglialatela A, Cai K, Huang JW, Leuzzi G, Cuella-Martin R, Palacios A, Gupta A, Egli D, Ciccia A*. Nat Commun 2019
• CRISPR-Mediated Base Editing Enables Efficient Disruption of Eukaryotic Genes through Induction of STOP Codons. Billon P, Bryant EE, Joseph SA, Nambiar TS, Hayward SB, Rothstein R, Ciccia A. Mol Cell 2017