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

Francesco Aulicino; Martin Pelosse; Christine Toelzer; Julien M Capin; Parisa Meysami; Mark S Dillingham; Christiane H Berger-Schaffitzel; Moin Saleem; Gavin I Welsh; Imre Berger; al et

doi:10.1093/nar/gkac587

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

Francesco Aulicino^*, Martin Pelosse, Christine Toelzer, Julien M Capin, Parisa Meysami, Mark S Dillingham, Christiane H Berger-Schaffitzel, Moin Saleem, Gavin I Welsh, Imre Berger^*, al et

^*Corresponding author for this work

Research output: Contribution to journal › Article (Academic Journal) › peer-review

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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 language	English
Pages (from-to)	7783–7799
Number of pages	17
Journal	Nucleic Acids Research
Volume	50
Issue number	13
Early online date	8 Jul 2022
DOIs	https://doi.org/10.1093/nar/gkac587
Publication status	E-pub ahead of print - 8 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.

Research Groups and Themes

Bristol BioDesign Institute
BrisSynBio
Max Planck Bristol

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Aulicino, F., Pelosse, M., Toelzer, C., Capin, J. M., Meysami, P., Dillingham, M. S., Berger-Schaffitzel, C. H., Saleem, M., Welsh, G. I., Berger, I., & et, A. (2022). Highly efficient CRISPR-mediated large DNA docking and multiplexed prime editing using a single baculovirus. Nucleic Acids Research, 50(13), 7783–7799. Advance online publication. https://doi.org/10.1093/nar/gkac587

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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.",

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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: {\textcopyright} The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.",

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AU - Aulicino, Francesco

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AU - Toelzer, Christine

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AU - Meysami, Parisa

AU - Dillingham, Mark S

AU - Berger-Schaffitzel, Christiane H

AU - Saleem, Moin

AU - Welsh, Gavin I

AU - Berger, Imre

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N1 - 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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N2 - 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.

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DO - 10.1093/nar/gkac587

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JO - Nucleic Acids Research

JF - Nucleic Acids Research

IS - 13

ER -

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

Abstract

Bibliographical note

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