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

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 Capin, Erwin Ilegems, Parisa Meysami, Ruth Rollarson, Per-Olof Berggren, Mark Simon Dillingham, Christiane Schaffitzel, Moin A Saleem, Gavin I Welsh, Imre Berger

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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	Published (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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10.1093/nar/gkac587

gkac587Final published version, 5.88 MBLicence: CC BY

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Aulicino, F., Pelosse, M., Toelzer, C., Capin, J., Ilegems, E., Meysami, P., Rollarson, R., Berggren, P.-O., Dillingham, M. S., Schaffitzel, C., Saleem, M. A., Welsh, G. I., & Berger, I. (2022). Highly efficient CRISPR-mediated large DNA docking and multiplexed prime editing using a single baculovirus. Nucleic Acids Research, 50(13), 7783-7799. 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.",

author = "Francesco Aulicino and Martin Pelosse and Christine Toelzer and Julien Capin and Erwin Ilegems and Parisa Meysami and Ruth Rollarson and Per-Olof Berggren and Dillingham, {Mark Simon} and Christiane Schaffitzel and Saleem, {Moin A} and Welsh, {Gavin I} and Imre Berger",

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Aulicino, F, Pelosse, M, Toelzer, C, Capin, J, Ilegems, E, Meysami, P, Rollarson, R, Berggren, P-O, Dillingham, MS, Schaffitzel, C, Saleem, MA, Welsh, GI & Berger, I 2022, 'Highly efficient CRISPR-mediated large DNA docking and multiplexed prime editing using a single baculovirus', Nucleic Acids Research, vol. 50, no. 13, pp. 7783-7799. https://doi.org/10.1093/nar/gkac587

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AU - Pelosse, Martin

AU - Toelzer, Christine

AU - Capin, Julien

AU - Ilegems, Erwin

AU - Meysami, Parisa

AU - Rollarson, Ruth

AU - Berggren, Per-Olof

AU - Dillingham, Mark Simon

AU - Schaffitzel, Christiane

AU - Saleem, Moin A

AU - Welsh, Gavin I

AU - Berger, Imre

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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Y1 - 2022/7/22

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

Abstract

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