CRISPRI-based high-throughput functional genomic approaches for use in mycobacteria
| dc.contributor.advisor | Warner, Digby | |
| dc.contributor.author | De Wet, Timothy | |
| dc.date.accessioned | 2022-01-31T07:50:42Z | |
| dc.date.available | 2022-01-31T07:50:42Z | |
| dc.date.issued | 2021 | |
| dc.date.updated | 2022-01-26T12:53:33Z | |
| dc.description.abstract | In the 20 years since the pioneering publication of the genome of Mycobacterium tuberculosis, significant efforts have been made to complete functional annotation of the genome. However, these efforts have generally been performed on a single-gene basis, ensuring slow progress and leaving large portions of the genome unannotated. High-throughput approaches to understanding the functional genome, such as transposon-insertion sequencing, have been developed and applied to mycobacteria in a variety of conditions; however, they have several limitations, particularly in their ability to study genes essential for viability. The recent optimisation of inducible CRISPR-interference for mycobacteria offers the potential to expand the high-throughput functional genomic toolkit. This thesis utilises CRISPR-interference for the development and validation of two high-throughput functional genomic approaches in the model mycobacterium M. smegmatis. The first approach combines large-scale pooled oligonucleotide synthesis and nextgeneration sequencing, and is termed CRISPRi-Seq. A pooled library of 11 367 mutants, targeting 2 385 M. smegmatis genes with M. tuberculosis homologues, was constructed and used to infer gene essentialities which were compared with corresponding predictions from transposon-insertion sequencing data. This process validated the CRISPRi-Seq technique and identified practical considerations for its future use. The second approach utilises data derived from CRISPRi-Seq to create an arrayed library of 263 individual M. smegmatis inducible CRISPRi mutants targeting essential genes. This library is applied to a quantitative imaging pipeline to produce detailed data-driven profiles of the morphological impact of essential gene suppression. These morphological profiles are used to statistically predict genetic function, as well as antimicrobial mechanism-of-action. The two novel approaches developed in this work represent valuable technical advances and produce large datasets of functional genomic data which are available interactively online. Taken individually, or in combination, these methodologies can be utilised to increase fundamental understanding of mycobacteria, including the pathogenic M. tuberculosis | |
| dc.identifier.apacitation | De Wet, T. (2021). <i>CRISPRI-based high-throughput functional genomic approaches for use in mycobacteria</i>. (). ,Faculty of Health Sciences ,Division of Medical Microbiology. Retrieved from http://hdl.handle.net/11427/35612 | en_ZA |
| dc.identifier.chicagocitation | De Wet, Timothy. <i>"CRISPRI-based high-throughput functional genomic approaches for use in mycobacteria."</i> ., ,Faculty of Health Sciences ,Division of Medical Microbiology, 2021. http://hdl.handle.net/11427/35612 | en_ZA |
| dc.identifier.citation | De Wet, T. 2021. CRISPRI-based high-throughput functional genomic approaches for use in mycobacteria. . ,Faculty of Health Sciences ,Division of Medical Microbiology. http://hdl.handle.net/11427/35612 | en_ZA |
| dc.identifier.ris | TY - Doctoral Thesis AU - De Wet, Timothy AB - In the 20 years since the pioneering publication of the genome of Mycobacterium tuberculosis, significant efforts have been made to complete functional annotation of the genome. However, these efforts have generally been performed on a single-gene basis, ensuring slow progress and leaving large portions of the genome unannotated. High-throughput approaches to understanding the functional genome, such as transposon-insertion sequencing, have been developed and applied to mycobacteria in a variety of conditions; however, they have several limitations, particularly in their ability to study genes essential for viability. The recent optimisation of inducible CRISPR-interference for mycobacteria offers the potential to expand the high-throughput functional genomic toolkit. This thesis utilises CRISPR-interference for the development and validation of two high-throughput functional genomic approaches in the model mycobacterium M. smegmatis. The first approach combines large-scale pooled oligonucleotide synthesis and nextgeneration sequencing, and is termed CRISPRi-Seq. A pooled library of 11 367 mutants, targeting 2 385 M. smegmatis genes with M. tuberculosis homologues, was constructed and used to infer gene essentialities which were compared with corresponding predictions from transposon-insertion sequencing data. This process validated the CRISPRi-Seq technique and identified practical considerations for its future use. The second approach utilises data derived from CRISPRi-Seq to create an arrayed library of 263 individual M. smegmatis inducible CRISPRi mutants targeting essential genes. This library is applied to a quantitative imaging pipeline to produce detailed data-driven profiles of the morphological impact of essential gene suppression. These morphological profiles are used to statistically predict genetic function, as well as antimicrobial mechanism-of-action. The two novel approaches developed in this work represent valuable technical advances and produce large datasets of functional genomic data which are available interactively online. Taken individually, or in combination, these methodologies can be utilised to increase fundamental understanding of mycobacteria, including the pathogenic M. tuberculosis DA - 2021_ DB - OpenUCT DP - University of Cape Town KW - Medical Microbiology LK - https://open.uct.ac.za PY - 2021 T1 - CRISPRI-based high-throughput functional genomic approaches for use in mycobacteria TI - CRISPRI-based high-throughput functional genomic approaches for use in mycobacteria UR - http://hdl.handle.net/11427/35612 ER - | en_ZA |
| dc.identifier.uri | http://hdl.handle.net/11427/35612 | |
| dc.identifier.vancouvercitation | De Wet T. CRISPRI-based high-throughput functional genomic approaches for use in mycobacteria. []. ,Faculty of Health Sciences ,Division of Medical Microbiology, 2021 [cited yyyy month dd]. Available from: http://hdl.handle.net/11427/35612 | en_ZA |
| dc.language.rfc3066 | eng | |
| dc.publisher.department | Division of Medical Microbiology | |
| dc.publisher.faculty | Faculty of Health Sciences | |
| dc.subject | Medical Microbiology | |
| dc.title | CRISPRI-based high-throughput functional genomic approaches for use in mycobacteria | |
| dc.type | Doctoral Thesis | |
| dc.type.qualificationlevel | Doctoral | |
| dc.type.qualificationlevel | PhD |