Evaluating the genotypic and phenotypic stability of CRISPRi essential gene mutants in Burkholderia cenocepacia K56-2
| dc.contributor.author | Batun Hernandez, Zayra Yael | |
| dc.contributor.examiningcommittee | Gerstein, Aleeza (Microbiology) | |
| dc.contributor.examiningcommittee | Budisa, Nediljko (Chemistry) | |
| dc.contributor.supervisor | Cardona, Silvia | |
| dc.date.accessioned | 2025-02-10T16:44:57Z | |
| dc.date.available | 2025-02-10T16:44:57Z | |
| dc.date.issued | 2025-01-31 | |
| dc.date.submitted | 2025-01-31T18:28:29Z | en_US |
| dc.degree.discipline | Microbiology | |
| dc.degree.level | Master of Science (M.Sc.) | |
| dc.description.abstract | Essential genes encode the processes required for cell growth and survival within specific environments. Understanding the products of essential genes is important for antibiotic discovery, as many essential genes encode the targets of antibiotics. Also, controlling the products of essential genes is a potential approach to reversibly inhibit growth while exploiting beneficial traits. A genetic tool to investigate essential gene function by controlling gene expression is CRISPR interference (CRISPRi). This tool comprises a nuclease-inactive Cas9 (dCas9) and a synthetic guide RNA (sgRNA), forming a ribonucleoprotein complex that binds to the target gene and sterically blocks transcription. Previously, we constructed a CRISPRi system in strains of the Burkholderia cepacia complex, opportunistic bacterial pathogens with biotechnological potential. Our CRISPRi system uses a chromosomally encoded, rhamnose-inducible dCas9 and a sgRNA constitutively expressed from a plasmid. We and others have observed that CRISPRi mutants spontaneously lose their conditional growth phenotype (CGP), hampering their potential use in controlling bacterial growth. To understand this phenotypic reversion, we performed experimental evolution experiments in selected 15 CRISPRi mutants under dCas9-inducing and non-inducing conditions. At time points, we performed targeted gene repression by RT-qPCR and whole genome sequencing (WGS) in parental and evolved mutants. While analysis across generations revealed diverse CGPs, all mutants reverted to wild-type growth phenotypes. K-medoids clustering identified 3 clusters with different relationships between essential gene functions and the CGP. Under consistent inducing conditions, Cluster 1 exhibited severe gene repression of 78-fold, dropping to 3-fold. Cluster 2 decreased from 46-fold to 0.5-fold, whereas Cluster 3 demonstrated wide diversity in gene repression. There was no evidence of single nucleotide polymorphisms (SNPs) in the dCas9, rhamnose system regions, or the encoded sgRNAs maintained on a plasmid. However, WGS identified SNPs in a porin and a LysR-type global regulator. Immunoblot analysis confirmed the absence of dCas9, indicating a lack of expression possibly due to porin malfunction affecting rhamnose uptake or protein degradation caused by unregulated proteases. Together, while the CRISPRi machinery is genetically stable, compensatory mutations compromised its function at longer times. Our work highlights the strengths and limitations of CRISPRi for extended control of bacterial growth. | |
| dc.description.note | May 2025 | |
| dc.description.sponsorship | Mitacs Globalink Graduate Fellowship (2021-2022) Research Manitoba - Natural Sciences & Engineering (2023-2024) | |
| dc.identifier.uri | http://hdl.handle.net/1993/38866 | |
| dc.language.iso | eng | |
| dc.subject | CRISPR interference (CRISPRi) | |
| dc.subject | Burkholderia | |
| dc.subject | Essential genes | |
| dc.subject | Bacterial genetics | |
| dc.subject | Conditional growth phenotype | |
| dc.title | Evaluating the genotypic and phenotypic stability of CRISPRi essential gene mutants in Burkholderia cenocepacia K56-2 | |
| local.subject.manitoba | no | |
| oaire.awardNumber | RGPIN-2022-04119 | |
| oaire.awardTitle | Synthetic Biology Tools to unleash the Biotechnological Potential of the genus Burkholderia | |
| oaire.awardURI | https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=760957 | |
| project.funder.name | Natural Sciences and Engineering Research Council of Canada (NSERC) |