Induced protein expression in Leptospira spp. and its application to CRISPR/Cas9 mutant generation
Abstract Expanding the genetic toolkit for Leptospira spp. is a crucial step toward advancing our understanding of the biology and virulence of these atypical bacteria. Pathogenic Leptospira are responsible for over 1 million human leptospirosis cases annually and significantly impact domestic anima...
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Nature Portfolio
2025-02-01
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| Online Access: | https://doi.org/10.1038/s41598-025-88633-w |
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| author | L. G. V. Fernandes A. L. T. O. Nascimento J. E. Nally |
| author_facet | L. G. V. Fernandes A. L. T. O. Nascimento J. E. Nally |
| author_sort | L. G. V. Fernandes |
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| description | Abstract Expanding the genetic toolkit for Leptospira spp. is a crucial step toward advancing our understanding of the biology and virulence of these atypical bacteria. Pathogenic Leptospira are responsible for over 1 million human leptospirosis cases annually and significantly impact domestic animals. Bovine leptospirosis causes substantial financial losses due to abortion, stillbirths, and suboptimal reproductive performance. The advent of the CRISPR/Cas9 system has marked a turning point in genetic manipulation, with applications across multiple Leptospira species. However, incorporating controlled protein expression into existing genetic tools could further expand their utility. We developed and demonstrated the functionality of IPTG-inducible heterologous protein expression in Leptospira spp. This system was applied for regulated expression of dead Cas9 (dCas9) to generate knockdown mutants, and Cas9 to produce knockout mutants by inducing double-strand breaks (DSB) into desired targets. IPTG-induced dCas9 expression enabled validation of essential genes and non-coding RNAs. Additionally, IPTG-controlled Cas9 expression combined with a constitutive non-homologous end-joining (NHEJ) system allowed for successful recovery of knockout mutants, even in the absence of IPTG. These newly controlled protein expression systems will advance studies on the basic biology and virulence of Leptospira, as well as facilitate knockout mutant generation for improved veterinary vaccines. |
| format | Article |
| id | doaj-art-07549fe764374c6eac75ebb72877c295 |
| institution | Kabale University |
| issn | 2045-2322 |
| language | English |
| publishDate | 2025-02-01 |
| publisher | Nature Portfolio |
| record_format | Article |
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| spelling | doaj-art-07549fe764374c6eac75ebb72877c2952025-02-09T12:34:33ZengNature PortfolioScientific Reports2045-23222025-02-0115111310.1038/s41598-025-88633-wInduced protein expression in Leptospira spp. and its application to CRISPR/Cas9 mutant generationL. G. V. Fernandes0A. L. T. O. Nascimento1J. E. Nally2Infectious Bacterial Diseases Research Unit, USDA Agricultural Research Service, National Animal Disease CenterLaboratório de Desenvolvimento de Vacinas, Instituto ButantanInfectious Bacterial Diseases Research Unit, USDA Agricultural Research Service, National Animal Disease CenterAbstract Expanding the genetic toolkit for Leptospira spp. is a crucial step toward advancing our understanding of the biology and virulence of these atypical bacteria. Pathogenic Leptospira are responsible for over 1 million human leptospirosis cases annually and significantly impact domestic animals. Bovine leptospirosis causes substantial financial losses due to abortion, stillbirths, and suboptimal reproductive performance. The advent of the CRISPR/Cas9 system has marked a turning point in genetic manipulation, with applications across multiple Leptospira species. However, incorporating controlled protein expression into existing genetic tools could further expand their utility. We developed and demonstrated the functionality of IPTG-inducible heterologous protein expression in Leptospira spp. This system was applied for regulated expression of dead Cas9 (dCas9) to generate knockdown mutants, and Cas9 to produce knockout mutants by inducing double-strand breaks (DSB) into desired targets. IPTG-induced dCas9 expression enabled validation of essential genes and non-coding RNAs. Additionally, IPTG-controlled Cas9 expression combined with a constitutive non-homologous end-joining (NHEJ) system allowed for successful recovery of knockout mutants, even in the absence of IPTG. These newly controlled protein expression systems will advance studies on the basic biology and virulence of Leptospira, as well as facilitate knockout mutant generation for improved veterinary vaccines.https://doi.org/10.1038/s41598-025-88633-wIPTGMutagenesisLeptospiraKnockdowndCas9Knockout |
| spellingShingle | L. G. V. Fernandes A. L. T. O. Nascimento J. E. Nally Induced protein expression in Leptospira spp. and its application to CRISPR/Cas9 mutant generation Scientific Reports IPTG Mutagenesis Leptospira Knockdown dCas9 Knockout |
| title | Induced protein expression in Leptospira spp. and its application to CRISPR/Cas9 mutant generation |
| title_full | Induced protein expression in Leptospira spp. and its application to CRISPR/Cas9 mutant generation |
| title_fullStr | Induced protein expression in Leptospira spp. and its application to CRISPR/Cas9 mutant generation |
| title_full_unstemmed | Induced protein expression in Leptospira spp. and its application to CRISPR/Cas9 mutant generation |
| title_short | Induced protein expression in Leptospira spp. and its application to CRISPR/Cas9 mutant generation |
| title_sort | induced protein expression in leptospira spp and its application to crispr cas9 mutant generation |
| topic | IPTG Mutagenesis Leptospira Knockdown dCas9 Knockout |
| url | https://doi.org/10.1038/s41598-025-88633-w |
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