Highly efficient CRISPR-mediated large DNA docking and multiplexed prime editing using a single baculovirus

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

    Research output: Contribution to journalArticlepeer-review

    14 Citations (Scopus)
    92 Downloads (Pure)

    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.

    Original languageEnglish
    Pages (from-to)7783-7799
    Number of pages17
    JournalNucleic Acids Research
    Volume50
    Issue number13
    Early online date8 Jul 2022
    DOIs
    Publication statusPublished (in print/issue) - 22 Jul 2022

    Bibliographical note

    Funding Information:
    European Research Council (ERC Advanced Grant) [DNA-DOCK, Project No. 834631]; GE Healthcare [Discovery Research Grant to I.B.]; BrisSynBio, a BBSRC/EPSRC Research Centre for Synthetic Biology at the University of Bristol [BB/L01386X/1 to I.B.]; Max Planck Centre for Minimal Biology, KRUK [KS RP 001 20190917 and Paed RP 003 20180928 to G.I.W. and R.R.]; MRC [MR/R013942/1 and MR/R003017/1 to M.A.S. and G.I.W.]. Funding for open access charge: University of Bristol.

    Publisher Copyright:
    © The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.

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