Streamlined and efficient genome editing in Cupriavidus necator H16 using an optimised SIBR-Cas system

Simona Della Valle; Enrico Orsi; Sjoerd C.A. Creutzburg; Luc F.M. Jansen; Evangelia Niki Pentari; Chase L. Beisel; Harrison Steel; Pablo I. Nikel; Raymond H.J. Staals; Nico J. Claassens; John van der Oost; Wei E. Huang; Constantinos Patinios

doi:10.1016/j.tibtech.2025.02.006

Streamlined and efficient genome editing in Cupriavidus necator H16 using an optimised SIBR-Cas system

Simona Della Valle, Enrico Orsi, Sjoerd C.A. Creutzburg, Luc F.M. Jansen, Evangelia Niki Pentari, Chase L. Beisel, Harrison Steel, Pablo I. Nikel, Raymond H.J. Staals, Nico J. Claassens, John van der Oost, Wei E. Huang^*, Constantinos Patinios

^*Corresponding author for this work

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

Abstract

Cupriavidus necator H16 is a promising microbial platform strain for CO₂ valorisation. While C. necator is amenable to genome editing, existing tools are often inefficient or rely on lengthy protocols, hindering its rapid transition to industrial applications. In this study, we simplified and accelerated the genome editing pipeline for C. necator by harnessing the Self-splicing Intron-Based Riboswitch (SIBR) system. We used SIBR to tightly control and delay Cas9-based counterselection, achieving >80% editing efficiency at two genomic loci within 48 h after electroporation. To further increase the versatility of the genome editing toolbox, we upgraded SIBR to SIBR2.0 and used it to regulate the expression of Cas12a. SIBR2.0-Cas12a could mediate gene deletion in C. necator with ~70% editing efficiency. Overall, we streamlined the genome editing pipeline for C. necator, facilitating its potential role in the transition to a bio-based economy.

Original language	English
Number of pages	22
Journal	Trends in Biotechnology
DOIs	https://doi.org/10.1016/j.tibtech.2025.02.006
Publication status	E-pub ahead of print - 13 Mar 2025

Keywords

CRISPR-Cas
Cupriavidus necator H16
genome editing
non-model microbes
SIBR
SIBR2.0

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Della Valle, S., Orsi, E., Creutzburg, S. C. A., Jansen, L. F. M., Pentari, E. N., Beisel, C. L., Steel, H., Nikel, P. I., Staals, R. H. J., Claassens, N. J., van der Oost, J., Huang, W. E., & Patinios, C. (2025). Streamlined and efficient genome editing in Cupriavidus necator H16 using an optimised SIBR-Cas system. Trends in Biotechnology. Advance online publication. https://doi.org/10.1016/j.tibtech.2025.02.006

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title = "Streamlined and efficient genome editing in Cupriavidus necator H16 using an optimised SIBR-Cas system",

abstract = "Cupriavidus necator H16 is a promising microbial platform strain for CO2 valorisation. While C. necator is amenable to genome editing, existing tools are often inefficient or rely on lengthy protocols, hindering its rapid transition to industrial applications. In this study, we simplified and accelerated the genome editing pipeline for C. necator by harnessing the Self-splicing Intron-Based Riboswitch (SIBR) system. We used SIBR to tightly control and delay Cas9-based counterselection, achieving >80% editing efficiency at two genomic loci within 48 h after electroporation. To further increase the versatility of the genome editing toolbox, we upgraded SIBR to SIBR2.0 and used it to regulate the expression of Cas12a. SIBR2.0-Cas12a could mediate gene deletion in C. necator with ~70% editing efficiency. Overall, we streamlined the genome editing pipeline for C. necator, facilitating its potential role in the transition to a bio-based economy.",

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author = "{Della Valle}, Simona and Enrico Orsi and Creutzburg, {Sjoerd C.A.} and Jansen, {Luc F.M.} and Pentari, {Evangelia Niki} and Beisel, {Chase L.} and Harrison Steel and Nikel, {Pablo I.} and Staals, {Raymond H.J.} and Claassens, {Nico J.} and {van der Oost}, John and Huang, {Wei E.} and Constantinos Patinios",

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doi = "10.1016/j.tibtech.2025.02.006",

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AU - Della Valle, Simona

AU - Orsi, Enrico

AU - Creutzburg, Sjoerd C.A.

AU - Jansen, Luc F.M.

AU - Pentari, Evangelia Niki

AU - Beisel, Chase L.

AU - Steel, Harrison

AU - Nikel, Pablo I.

AU - Staals, Raymond H.J.

AU - Claassens, Nico J.

AU - van der Oost, John

AU - Huang, Wei E.

AU - Patinios, Constantinos

PY - 2025/3/13

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N2 - Cupriavidus necator H16 is a promising microbial platform strain for CO2 valorisation. While C. necator is amenable to genome editing, existing tools are often inefficient or rely on lengthy protocols, hindering its rapid transition to industrial applications. In this study, we simplified and accelerated the genome editing pipeline for C. necator by harnessing the Self-splicing Intron-Based Riboswitch (SIBR) system. We used SIBR to tightly control and delay Cas9-based counterselection, achieving >80% editing efficiency at two genomic loci within 48 h after electroporation. To further increase the versatility of the genome editing toolbox, we upgraded SIBR to SIBR2.0 and used it to regulate the expression of Cas12a. SIBR2.0-Cas12a could mediate gene deletion in C. necator with ~70% editing efficiency. Overall, we streamlined the genome editing pipeline for C. necator, facilitating its potential role in the transition to a bio-based economy.

AB - Cupriavidus necator H16 is a promising microbial platform strain for CO2 valorisation. While C. necator is amenable to genome editing, existing tools are often inefficient or rely on lengthy protocols, hindering its rapid transition to industrial applications. In this study, we simplified and accelerated the genome editing pipeline for C. necator by harnessing the Self-splicing Intron-Based Riboswitch (SIBR) system. We used SIBR to tightly control and delay Cas9-based counterselection, achieving >80% editing efficiency at two genomic loci within 48 h after electroporation. To further increase the versatility of the genome editing toolbox, we upgraded SIBR to SIBR2.0 and used it to regulate the expression of Cas12a. SIBR2.0-Cas12a could mediate gene deletion in C. necator with ~70% editing efficiency. Overall, we streamlined the genome editing pipeline for C. necator, facilitating its potential role in the transition to a bio-based economy.

KW - CRISPR-Cas

KW - Cupriavidus necator H16

KW - genome editing

KW - non-model microbes

KW - SIBR

KW - SIBR2.0

U2 - 10.1016/j.tibtech.2025.02.006

DO - 10.1016/j.tibtech.2025.02.006

M3 - Article

AN - SCOPUS:86000766324

SN - 0167-7799

JO - Trends in Biotechnology

JF - Trends in Biotechnology

ER -

Streamlined and efficient genome editing in Cupriavidus necator H16 using an optimised SIBR-Cas system

Abstract

Keywords

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