CRISPR/Cas9-mediated genomic insertion of functional genes into Lactiplantibacillus plantarum WCFS1
ABSTRACT Lactiplantibacillus plantarum, a natural inhabitant of the human body, is a promising candidate vehicle for vaccine delivery. An obstacle in developing bacterial delivery vehicles is generating a production strain that lacks antibiotic resistance genes and contains minimal foreign DNA. To d...
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| Language: | English |
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American Society for Microbiology
2025-02-01
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| Series: | Microbiology Spectrum |
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| Online Access: | https://journals.asm.org/doi/10.1128/spectrum.02025-24 |
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| author | Kamilla Wiull Lisa K. Haugen Vincent G. H. Eijsink Geir Mathiesen |
| author_facet | Kamilla Wiull Lisa K. Haugen Vincent G. H. Eijsink Geir Mathiesen |
| author_sort | Kamilla Wiull |
| collection | DOAJ |
| description | ABSTRACT Lactiplantibacillus plantarum, a natural inhabitant of the human body, is a promising candidate vehicle for vaccine delivery. An obstacle in developing bacterial delivery vehicles is generating a production strain that lacks antibiotic resistance genes and contains minimal foreign DNA. To deal with this obstacle, we have constructed a finetuned, inducible two-plasmid CRISPR/Cas9-system for chromosomal gene insertion in L. plantarum. The knock-in plasmid was designed with a cassette-like structure to simplify the insertion of target DNA and streamline the CRISPR/Cas9 genome editing, bringing it one step closer to becoming a routine procedure. We demonstrate that the system enables efficient insertion of expression cassettes for both inducible and constitutive production of a fluorescent reporter protein, mCherry, and for inducible production of the receptor-binding domain (RBD) of the SARS-CoV-2 virus. Two variants of RBD were successfully expressed, one directed to the cytoplasm and one directed to the cell surface. All the knock-in strains produced the target protein, although with lower yields than strains with plasmid-encoded expression.IMPORTANCEGenetic engineering of lactic acid bacteria, such as Lactiplantibacillus plantarum, has proven to be difficult. This study presents an inducible two-plasmid CRISPR/Cas9-system for inserting genes into the chromosome of Lactiplantibacillus plantarum. Our system successfully knock-in four expression cassettes varying in length from ~800–1,300 bp with high efficiency and insert an expression cassette encoding a SARS-CoV-2 antigen receptor-binding domain (RBD) with an anchor mediating surface display, which has not been achieved previously using CRISPR/Cas9. We demonstrate the production of the insertion genes. Importantly, the plasmid carrying the SgRNA, Cas9, and homology-directed repair template is designed for easy component exchange. These plasmids represent valuable contributions to the field as they could facilitate rapid CRISPR/Cas9 engineering of L. plantarum strains. |
| format | Article |
| id | doaj-art-9b304b027f2c49de992f5e0e00ed9f1f |
| institution | Kabale University |
| issn | 2165-0497 |
| language | English |
| publishDate | 2025-02-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | Microbiology Spectrum |
| spelling | doaj-art-9b304b027f2c49de992f5e0e00ed9f1f2025-02-04T14:03:41ZengAmerican Society for MicrobiologyMicrobiology Spectrum2165-04972025-02-0113210.1128/spectrum.02025-24CRISPR/Cas9-mediated genomic insertion of functional genes into Lactiplantibacillus plantarum WCFS1Kamilla Wiull0Lisa K. Haugen1Vincent G. H. Eijsink2Geir MathiesenFaculty of Chemistry, Biotechnology and Food Science, NMBU - Norwegian University of Life Sciences, Ås, NorwayFaculty of Chemistry, Biotechnology and Food Science, NMBU - Norwegian University of Life Sciences, Ås, NorwayFaculty of Chemistry, Biotechnology and Food Science, NMBU - Norwegian University of Life Sciences, Ås, NorwayABSTRACT Lactiplantibacillus plantarum, a natural inhabitant of the human body, is a promising candidate vehicle for vaccine delivery. An obstacle in developing bacterial delivery vehicles is generating a production strain that lacks antibiotic resistance genes and contains minimal foreign DNA. To deal with this obstacle, we have constructed a finetuned, inducible two-plasmid CRISPR/Cas9-system for chromosomal gene insertion in L. plantarum. The knock-in plasmid was designed with a cassette-like structure to simplify the insertion of target DNA and streamline the CRISPR/Cas9 genome editing, bringing it one step closer to becoming a routine procedure. We demonstrate that the system enables efficient insertion of expression cassettes for both inducible and constitutive production of a fluorescent reporter protein, mCherry, and for inducible production of the receptor-binding domain (RBD) of the SARS-CoV-2 virus. Two variants of RBD were successfully expressed, one directed to the cytoplasm and one directed to the cell surface. All the knock-in strains produced the target protein, although with lower yields than strains with plasmid-encoded expression.IMPORTANCEGenetic engineering of lactic acid bacteria, such as Lactiplantibacillus plantarum, has proven to be difficult. This study presents an inducible two-plasmid CRISPR/Cas9-system for inserting genes into the chromosome of Lactiplantibacillus plantarum. Our system successfully knock-in four expression cassettes varying in length from ~800–1,300 bp with high efficiency and insert an expression cassette encoding a SARS-CoV-2 antigen receptor-binding domain (RBD) with an anchor mediating surface display, which has not been achieved previously using CRISPR/Cas9. We demonstrate the production of the insertion genes. Importantly, the plasmid carrying the SgRNA, Cas9, and homology-directed repair template is designed for easy component exchange. These plasmids represent valuable contributions to the field as they could facilitate rapid CRISPR/Cas9 engineering of L. plantarum strains.https://journals.asm.org/doi/10.1128/spectrum.02025-24genome editinggene insertionLactiplantibacillus plantarumCRISPRsurface display |
| spellingShingle | Kamilla Wiull Lisa K. Haugen Vincent G. H. Eijsink Geir Mathiesen CRISPR/Cas9-mediated genomic insertion of functional genes into Lactiplantibacillus plantarum WCFS1 Microbiology Spectrum genome editing gene insertion Lactiplantibacillus plantarum CRISPR surface display |
| title | CRISPR/Cas9-mediated genomic insertion of functional genes into Lactiplantibacillus plantarum WCFS1 |
| title_full | CRISPR/Cas9-mediated genomic insertion of functional genes into Lactiplantibacillus plantarum WCFS1 |
| title_fullStr | CRISPR/Cas9-mediated genomic insertion of functional genes into Lactiplantibacillus plantarum WCFS1 |
| title_full_unstemmed | CRISPR/Cas9-mediated genomic insertion of functional genes into Lactiplantibacillus plantarum WCFS1 |
| title_short | CRISPR/Cas9-mediated genomic insertion of functional genes into Lactiplantibacillus plantarum WCFS1 |
| title_sort | crispr cas9 mediated genomic insertion of functional genes into lactiplantibacillus plantarum wcfs1 |
| topic | genome editing gene insertion Lactiplantibacillus plantarum CRISPR surface display |
| url | https://journals.asm.org/doi/10.1128/spectrum.02025-24 |
| work_keys_str_mv | AT kamillawiull crisprcas9mediatedgenomicinsertionoffunctionalgenesintolactiplantibacillusplantarumwcfs1 AT lisakhaugen crisprcas9mediatedgenomicinsertionoffunctionalgenesintolactiplantibacillusplantarumwcfs1 AT vincentgheijsink crisprcas9mediatedgenomicinsertionoffunctionalgenesintolactiplantibacillusplantarumwcfs1 AT geirmathiesen crisprcas9mediatedgenomicinsertionoffunctionalgenesintolactiplantibacillusplantarumwcfs1 |