CRISPR/Cas12a-mediated gene silencing across diverse functional genes demonstrates single gene-specific spacer efficacy in Mycobacterium smegmatis
Abstract Background Tuberculosis, a persistent global health threat, necessitates a comprehensive understanding of the genes and pathways crucial for the survival and virulence of the causative pathogen, Mycobacterium tuberculosis. Working with M. tuberculosis (M.tb) presents significant challenges;...
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BMC
2025-02-01
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| Series: | Journal of Biological Engineering |
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| Online Access: | https://doi.org/10.1186/s13036-025-00490-3 |
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| author | Megha Sodani Chitra S. Misra Savita Kulkarni Devashish Rath |
| author_facet | Megha Sodani Chitra S. Misra Savita Kulkarni Devashish Rath |
| author_sort | Megha Sodani |
| collection | DOAJ |
| description | Abstract Background Tuberculosis, a persistent global health threat, necessitates a comprehensive understanding of the genes and pathways crucial for the survival and virulence of the causative pathogen, Mycobacterium tuberculosis. Working with M. tuberculosis (M.tb) presents significant challenges; therefore, the use of M. smegmatis as a surrogate system for conducting genetic studies of M.tb has proven to be highly valuable. Development of novel genetic tools to probe cellular processes accelerates the progress in the field of drug development and also helps in understanding the basic physiology of the bacterium. Results This study reports the successful implementation and evaluation of the CRISPR-Cas12a system for gene repression in Mycobacterium smegmatis, a surrogate for M. tuberculosis. We engineered a Cas12a-based CRISPR interference (CRISPRi) system and assessed its functionality. Targeting 45 genes with a single sgRNA per gene, we achieved efficient gene repression, leading to marked phenotypic changes. Each knockdown strain was evaluated individually for growth phenotypes, and a comparison of the results with the reported essential gene library probed with dCas9 demonstrated congruous results across diverse gene categories. The study shows that CRISPR/Cas12a system can be effectively utilised with a single gene specific target for efficient silencing of the gene and highlights the importance of subsequent growth assays required to evaluate the vulnerability of targeted gene silencing. Conclusion Our findings reveal the robustness and versatility of the dCas12a-CRISPRi system in M. smegmatis, providing a valuable tool for functional genomics research. This work showcases the potential of the dCas12a-CRISPRi system in investigating essential genes, enabling a deeper understanding of the biology and potential therapeutic targets in mycobacterium species. |
| format | Article |
| id | doaj-art-cf1b4b89eb10443aa43620db43ad9bfc |
| institution | DOAJ |
| issn | 1754-1611 |
| language | English |
| publishDate | 2025-02-01 |
| publisher | BMC |
| record_format | Article |
| series | Journal of Biological Engineering |
| spelling | doaj-art-cf1b4b89eb10443aa43620db43ad9bfc2025-08-20T03:04:16ZengBMCJournal of Biological Engineering1754-16112025-02-0119111410.1186/s13036-025-00490-3CRISPR/Cas12a-mediated gene silencing across diverse functional genes demonstrates single gene-specific spacer efficacy in Mycobacterium smegmatisMegha Sodani0Chitra S. Misra1Savita Kulkarni2Devashish Rath3Radiation Medicine Centre, Medical Group, Bhabha Atomic Research CentreApplied Genomics Section, Bio-Science Group, Bhabha Atomic Research CentreRadiation Medicine Centre, Medical Group, Bhabha Atomic Research CentreApplied Genomics Section, Bio-Science Group, Bhabha Atomic Research CentreAbstract Background Tuberculosis, a persistent global health threat, necessitates a comprehensive understanding of the genes and pathways crucial for the survival and virulence of the causative pathogen, Mycobacterium tuberculosis. Working with M. tuberculosis (M.tb) presents significant challenges; therefore, the use of M. smegmatis as a surrogate system for conducting genetic studies of M.tb has proven to be highly valuable. Development of novel genetic tools to probe cellular processes accelerates the progress in the field of drug development and also helps in understanding the basic physiology of the bacterium. Results This study reports the successful implementation and evaluation of the CRISPR-Cas12a system for gene repression in Mycobacterium smegmatis, a surrogate for M. tuberculosis. We engineered a Cas12a-based CRISPR interference (CRISPRi) system and assessed its functionality. Targeting 45 genes with a single sgRNA per gene, we achieved efficient gene repression, leading to marked phenotypic changes. Each knockdown strain was evaluated individually for growth phenotypes, and a comparison of the results with the reported essential gene library probed with dCas9 demonstrated congruous results across diverse gene categories. The study shows that CRISPR/Cas12a system can be effectively utilised with a single gene specific target for efficient silencing of the gene and highlights the importance of subsequent growth assays required to evaluate the vulnerability of targeted gene silencing. Conclusion Our findings reveal the robustness and versatility of the dCas12a-CRISPRi system in M. smegmatis, providing a valuable tool for functional genomics research. This work showcases the potential of the dCas12a-CRISPRi system in investigating essential genes, enabling a deeper understanding of the biology and potential therapeutic targets in mycobacterium species.https://doi.org/10.1186/s13036-025-00490-3CRISPRiCas12aMycobacteriumEssential geneGene silencing |
| spellingShingle | Megha Sodani Chitra S. Misra Savita Kulkarni Devashish Rath CRISPR/Cas12a-mediated gene silencing across diverse functional genes demonstrates single gene-specific spacer efficacy in Mycobacterium smegmatis Journal of Biological Engineering CRISPRi Cas12a Mycobacterium Essential gene Gene silencing |
| title | CRISPR/Cas12a-mediated gene silencing across diverse functional genes demonstrates single gene-specific spacer efficacy in Mycobacterium smegmatis |
| title_full | CRISPR/Cas12a-mediated gene silencing across diverse functional genes demonstrates single gene-specific spacer efficacy in Mycobacterium smegmatis |
| title_fullStr | CRISPR/Cas12a-mediated gene silencing across diverse functional genes demonstrates single gene-specific spacer efficacy in Mycobacterium smegmatis |
| title_full_unstemmed | CRISPR/Cas12a-mediated gene silencing across diverse functional genes demonstrates single gene-specific spacer efficacy in Mycobacterium smegmatis |
| title_short | CRISPR/Cas12a-mediated gene silencing across diverse functional genes demonstrates single gene-specific spacer efficacy in Mycobacterium smegmatis |
| title_sort | crispr cas12a mediated gene silencing across diverse functional genes demonstrates single gene specific spacer efficacy in mycobacterium smegmatis |
| topic | CRISPRi Cas12a Mycobacterium Essential gene Gene silencing |
| url | https://doi.org/10.1186/s13036-025-00490-3 |
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