Glow-in-the-Dark Infectious Disease Diagnostics Using CRISPR-Cas9-Based Split Luciferase Complementation

Harmen J. van der Veer; Eva A. van Aalen; Claire M.S. Michielsen; Eva T.L. Hanckmann; Jeroen Deckers; Marcel M.G.J. van Borren; Jacky Flipse; Anne J.M. Loonen; Joost P.H. Schoeber; Maarten Merkx

doi:10.1021/acscentsci.2c01467

Glow-in-the-Dark Infectious Disease Diagnostics Using CRISPR-Cas9-Based Split Luciferase Complementation

Harmen J. van der Veer, Eva A. van Aalen, Claire M.S. Michielsen, Eva T.L. Hanckmann, Jeroen Deckers, Marcel M.G.J. van Borren, Jacky Flipse, Anne J.M. Loonen, Joost P.H. Schoeber, Maarten Merkx (Corresponding author)

Onderzoeksoutput: Bijdrage aan tijdschrift › Tijdschriftartikel › Academic › peer review

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Nucleic acid detection methods based on CRISPR and isothermal amplification techniques show great potential for point-of-care diagnostic applications. However, most current methods rely on fluorescent or lateral flow assay readout, requiring external excitation or postamplification reaction transfer. Here, we developed a bioluminescent nucleic acid sensor (LUNAS) platform in which target dsDNA is sequence-specifically detected by a pair of dCas9-based probes mediating split NanoLuc luciferase complementation. LUNAS is easily integrated with recombinase polymerase amplification (RPA), providing attomolar sensitivity in a rapid one-pot assay. A calibrator luciferase is included for a robust ratiometric readout, enabling real-time monitoring of the RPA reaction using a simple digital camera. We designed an RT-RPA-LUNAS assay that allows SARS-CoV-2 RNA detection without the need for cumbersome RNA isolation and demonstrated its diagnostic performance for COVID-19 patient nasopharyngeal swab samples. Detection of SARS-CoV-2 from samples with viral RNA loads of ∼200 cp/μL was achieved within ∼20 min, showing that RPA-LUNAS is attractive for point-of-care infectious disease testing.

Originele taal-2	Engels
Pagina's (van-tot)	657-667
Aantal pagina's	11
Tijdschrift	ACS Central Science
Volume	9
Nummer van het tijdschrift	4
DOI's	https://doi.org/10.1021/acscentsci.2c01467
Status	Gepubliceerd - 26 apr. 2023

Bibliografische nota

Funding Information:
We especially thank the other members of the iGEM 2019 Eindhoven University of Technology team Jo-Anne Ewald, Noëlle Gerards, Anouk Marinus, Roy van Mierlo, Yvonne van Mil, Mandy Shao, and Chris Tomassen for their contribution to the iGEM project that lies at the base of this work. We thank Juliëtte Schaghecke (Fontys University of Applied Sciences, Eindhoven, The Netherlands) for her help in performing RT-ddPCR. We are thankful to the FreeGenes project for providing SARS-CoV-2 positive control plasmids. The following reagents were obtained through BEI Resources, NIAID, NIH: Genomic RNA from HCoV-OC43 (NR-52727), HCoV-229E (NR-52728), and SARS-CoV-2 (NR-52347). This work was supported by funding from the Dutch Research Council | Nationaal Regieorgaan Praktijkgericht Onderzoek SIA (NRPO-SIA) [RAAK.PRO02.066] and the Eindhoven University Fund [COVID-19 Engineering Fund].

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van der Veer, H. J., van Aalen, E. A., Michielsen, C. M. S., Hanckmann, E. T. L., Deckers, J., van Borren, M. M. G. J., Flipse, J., Loonen, A. J. M., Schoeber, J. P. H., & Merkx, M. (2023). Glow-in-the-Dark Infectious Disease Diagnostics Using CRISPR-Cas9-Based Split Luciferase Complementation. ACS Central Science, 9(4), 657-667. https://doi.org/10.1021/acscentsci.2c01467

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title = "Glow-in-the-Dark Infectious Disease Diagnostics Using CRISPR-Cas9-Based Split Luciferase Complementation",

abstract = "Nucleic acid detection methods based on CRISPR and isothermal amplification techniques show great potential for point-of-care diagnostic applications. However, most current methods rely on fluorescent or lateral flow assay readout, requiring external excitation or postamplification reaction transfer. Here, we developed a bioluminescent nucleic acid sensor (LUNAS) platform in which target dsDNA is sequence-specifically detected by a pair of dCas9-based probes mediating split NanoLuc luciferase complementation. LUNAS is easily integrated with recombinase polymerase amplification (RPA), providing attomolar sensitivity in a rapid one-pot assay. A calibrator luciferase is included for a robust ratiometric readout, enabling real-time monitoring of the RPA reaction using a simple digital camera. We designed an RT-RPA-LUNAS assay that allows SARS-CoV-2 RNA detection without the need for cumbersome RNA isolation and demonstrated its diagnostic performance for COVID-19 patient nasopharyngeal swab samples. Detection of SARS-CoV-2 from samples with viral RNA loads of ∼200 cp/μL was achieved within ∼20 min, showing that RPA-LUNAS is attractive for point-of-care infectious disease testing.",

author = "{van der Veer}, {Harmen J.} and {van Aalen}, {Eva A.} and Michielsen, {Claire M.S.} and Hanckmann, {Eva T.L.} and Jeroen Deckers and {van Borren}, {Marcel M.G.J.} and Jacky Flipse and Loonen, {Anne J.M.} and Schoeber, {Joost P.H.} and Maarten Merkx",

note = "Funding Information: We especially thank the other members of the iGEM 2019 Eindhoven University of Technology team Jo-Anne Ewald, No{\"e}lle Gerards, Anouk Marinus, Roy van Mierlo, Yvonne van Mil, Mandy Shao, and Chris Tomassen for their contribution to the iGEM project that lies at the base of this work. We thank Juli{\"e}tte Schaghecke (Fontys University of Applied Sciences, Eindhoven, The Netherlands) for her help in performing RT-ddPCR. We are thankful to the FreeGenes project for providing SARS-CoV-2 positive control plasmids. The following reagents were obtained through BEI Resources, NIAID, NIH: Genomic RNA from HCoV-OC43 (NR-52727), HCoV-229E (NR-52728), and SARS-CoV-2 (NR-52347). This work was supported by funding from the Dutch Research Council | Nationaal Regieorgaan Praktijkgericht Onderzoek SIA (NRPO-SIA) [RAAK.PRO02.066] and the Eindhoven University Fund [COVID-19 Engineering Fund]. ",

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AU - Hanckmann, Eva T.L.

AU - Deckers, Jeroen

AU - van Borren, Marcel M.G.J.

AU - Flipse, Jacky

AU - Loonen, Anne J.M.

AU - Schoeber, Joost P.H.

AU - Merkx, Maarten

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N2 - Nucleic acid detection methods based on CRISPR and isothermal amplification techniques show great potential for point-of-care diagnostic applications. However, most current methods rely on fluorescent or lateral flow assay readout, requiring external excitation or postamplification reaction transfer. Here, we developed a bioluminescent nucleic acid sensor (LUNAS) platform in which target dsDNA is sequence-specifically detected by a pair of dCas9-based probes mediating split NanoLuc luciferase complementation. LUNAS is easily integrated with recombinase polymerase amplification (RPA), providing attomolar sensitivity in a rapid one-pot assay. A calibrator luciferase is included for a robust ratiometric readout, enabling real-time monitoring of the RPA reaction using a simple digital camera. We designed an RT-RPA-LUNAS assay that allows SARS-CoV-2 RNA detection without the need for cumbersome RNA isolation and demonstrated its diagnostic performance for COVID-19 patient nasopharyngeal swab samples. Detection of SARS-CoV-2 from samples with viral RNA loads of ∼200 cp/μL was achieved within ∼20 min, showing that RPA-LUNAS is attractive for point-of-care infectious disease testing.

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Glow-in-the-Dark Infectious Disease Diagnostics Using CRISPR-Cas9-Based Split Luciferase Complementation

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