Highly Efficient CRISPR-mediated Large DNA Docking and Multiplexed Prime Editing Using a Single Baculovirus

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 2022 Jul 8

PMID 35801912

Authors

Francesco Aulicino

Martin Pelosse

Christine Toelzer

Julien Capin

Erwin Ilegems

Parisa Meysami

Ruth Rollarson

Per-Olof Berggren

Mark Simon Dillingham

Christiane Schaffitzel

Moin A Saleem

Gavin I Welsh

Imre Berger

Affiliations

Soon will be listed here.

Abstract

CRISPR-based precise gene-editing requires simultaneous delivery of multiple components into living cells, rapidly exceeding the cargo capacity of traditional viral vector systems. This challenge represents a major roadblock to genome engineering applications. Here we exploit the unmatched heterologous DNA cargo capacity of baculovirus to resolve this bottleneck in human cells. By encoding Cas9, sgRNA and Donor DNAs on a single, rapidly assembled baculoviral vector, we achieve with up to 30% efficacy whole-exon replacement in the intronic β-actin (ACTB) locus, including site-specific docking of very large DNA payloads. We use our approach to rescue wild-type podocin expression in steroid-resistant nephrotic syndrome (SRNS) patient derived podocytes. We demonstrate single baculovirus vectored delivery of single and multiplexed prime-editing toolkits, achieving up to 100% cleavage-free DNA search-and-replace interventions without detectable indels. Taken together, we provide a versatile delivery platform for single base to multi-gene level genome interventions, addressing the currently unmet need for a powerful delivery system accommodating current and future CRISPR technologies without the burden of limited cargo capacity.

Citing Articles

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