A Dual-Plasmid-Based CRISPR/Cas9-Mediated Strategy Enables Targeted Editing of pH Regulatory Gene <i>pacC</i> in a Clinical Isolate of <i>Trichophyton rubrum</i>
<i>Trichophyton rubrum</i> is the most prevalent causative agent responsible for 80–90% of all known superficial fungal infections in humans, worldwide. Limited available methods for genetic manipulations have been one of the major bottlenecks in understanding relevant molecular mechanis...
| Main Authors: | , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2022-11-01
|
| Series: | Journal of Fungi |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2309-608X/8/12/1241 |
| _version_ | 1797456847554215936 |
|---|---|
| author | Sanchita Sanchaya Dey Sivaprakash Ramalingam Bhupesh Taneja |
| author_facet | Sanchita Sanchaya Dey Sivaprakash Ramalingam Bhupesh Taneja |
| author_sort | Sanchita Sanchaya Dey |
| collection | DOAJ |
| description | <i>Trichophyton rubrum</i> is the most prevalent causative agent responsible for 80–90% of all known superficial fungal infections in humans, worldwide. Limited available methods for genetic manipulations have been one of the major bottlenecks in understanding relevant molecular mechanisms of disease pathogenesis in <i>T. rubrum</i>. Here, a dual-plasmid-based CRISPR/Cas9 strategy to edit pH regulatory transcription factor, <i>pacC</i>, of a clinical isolate of <i>T. rubrum</i> by non-homologous end joining (NHEJ) repair is presented. A cas9–eGFP fusion that aids pre-screening of primary transformants through detection of GFP fluorescence is expressed from one plasmid while target-specific sgRNA from the other brings about mutagenesis of <i>pacC</i> with an overall efficiency of 33.8–37.3%. The mutants had reduced transcript levels of <i>pacC</i> at both acidic and alkaline pH with several morphological abnormalities. We believe this dual-plasmid-based CRISPR/Cas9 strategy will aid functional genomics studies, especially in non-lab-adapted clinical strains of <i>T. rubrum</i>. |
| first_indexed | 2024-03-09T16:13:43Z |
| format | Article |
| id | doaj.art-3b66d1aff9f845f2b5f2901c7e28c9d2 |
| institution | Directory Open Access Journal |
| issn | 2309-608X |
| language | English |
| last_indexed | 2024-03-09T16:13:43Z |
| publishDate | 2022-11-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Journal of Fungi |
| spelling | doaj.art-3b66d1aff9f845f2b5f2901c7e28c9d22023-11-24T15:59:07ZengMDPI AGJournal of Fungi2309-608X2022-11-01812124110.3390/jof8121241A Dual-Plasmid-Based CRISPR/Cas9-Mediated Strategy Enables Targeted Editing of pH Regulatory Gene <i>pacC</i> in a Clinical Isolate of <i>Trichophyton rubrum</i>Sanchita Sanchaya Dey0Sivaprakash Ramalingam1Bhupesh Taneja2CSIR-Institute of Genomics and Integrative Biology (CSIR-IGIB), New Delhi 110025, IndiaCSIR-Institute of Genomics and Integrative Biology (CSIR-IGIB), New Delhi 110025, IndiaCSIR-Institute of Genomics and Integrative Biology (CSIR-IGIB), New Delhi 110025, India<i>Trichophyton rubrum</i> is the most prevalent causative agent responsible for 80–90% of all known superficial fungal infections in humans, worldwide. Limited available methods for genetic manipulations have been one of the major bottlenecks in understanding relevant molecular mechanisms of disease pathogenesis in <i>T. rubrum</i>. Here, a dual-plasmid-based CRISPR/Cas9 strategy to edit pH regulatory transcription factor, <i>pacC</i>, of a clinical isolate of <i>T. rubrum</i> by non-homologous end joining (NHEJ) repair is presented. A cas9–eGFP fusion that aids pre-screening of primary transformants through detection of GFP fluorescence is expressed from one plasmid while target-specific sgRNA from the other brings about mutagenesis of <i>pacC</i> with an overall efficiency of 33.8–37.3%. The mutants had reduced transcript levels of <i>pacC</i> at both acidic and alkaline pH with several morphological abnormalities. We believe this dual-plasmid-based CRISPR/Cas9 strategy will aid functional genomics studies, especially in non-lab-adapted clinical strains of <i>T. rubrum</i>.https://www.mdpi.com/2309-608X/8/12/1241dermatophytesCRISPR/Cas9NHEJ repair<i>Trichophyton rubrum</i>genome editing<i>pacC</i> |
| spellingShingle | Sanchita Sanchaya Dey Sivaprakash Ramalingam Bhupesh Taneja A Dual-Plasmid-Based CRISPR/Cas9-Mediated Strategy Enables Targeted Editing of pH Regulatory Gene <i>pacC</i> in a Clinical Isolate of <i>Trichophyton rubrum</i> Journal of Fungi dermatophytes CRISPR/Cas9 NHEJ repair <i>Trichophyton rubrum</i> genome editing <i>pacC</i> |
| title | A Dual-Plasmid-Based CRISPR/Cas9-Mediated Strategy Enables Targeted Editing of pH Regulatory Gene <i>pacC</i> in a Clinical Isolate of <i>Trichophyton rubrum</i> |
| title_full | A Dual-Plasmid-Based CRISPR/Cas9-Mediated Strategy Enables Targeted Editing of pH Regulatory Gene <i>pacC</i> in a Clinical Isolate of <i>Trichophyton rubrum</i> |
| title_fullStr | A Dual-Plasmid-Based CRISPR/Cas9-Mediated Strategy Enables Targeted Editing of pH Regulatory Gene <i>pacC</i> in a Clinical Isolate of <i>Trichophyton rubrum</i> |
| title_full_unstemmed | A Dual-Plasmid-Based CRISPR/Cas9-Mediated Strategy Enables Targeted Editing of pH Regulatory Gene <i>pacC</i> in a Clinical Isolate of <i>Trichophyton rubrum</i> |
| title_short | A Dual-Plasmid-Based CRISPR/Cas9-Mediated Strategy Enables Targeted Editing of pH Regulatory Gene <i>pacC</i> in a Clinical Isolate of <i>Trichophyton rubrum</i> |
| title_sort | dual plasmid based crispr cas9 mediated strategy enables targeted editing of ph regulatory gene i pacc i in a clinical isolate of i trichophyton rubrum i |
| topic | dermatophytes CRISPR/Cas9 NHEJ repair <i>Trichophyton rubrum</i> genome editing <i>pacC</i> |
| url | https://www.mdpi.com/2309-608X/8/12/1241 |
| work_keys_str_mv | AT sanchitasanchayadey adualplasmidbasedcrisprcas9mediatedstrategyenablestargetededitingofphregulatorygeneipacciinaclinicalisolateofitrichophytonrubrumi AT sivaprakashramalingam adualplasmidbasedcrisprcas9mediatedstrategyenablestargetededitingofphregulatorygeneipacciinaclinicalisolateofitrichophytonrubrumi AT bhupeshtaneja adualplasmidbasedcrisprcas9mediatedstrategyenablestargetededitingofphregulatorygeneipacciinaclinicalisolateofitrichophytonrubrumi AT sanchitasanchayadey dualplasmidbasedcrisprcas9mediatedstrategyenablestargetededitingofphregulatorygeneipacciinaclinicalisolateofitrichophytonrubrumi AT sivaprakashramalingam dualplasmidbasedcrisprcas9mediatedstrategyenablestargetededitingofphregulatorygeneipacciinaclinicalisolateofitrichophytonrubrumi AT bhupeshtaneja dualplasmidbasedcrisprcas9mediatedstrategyenablestargetededitingofphregulatorygeneipacciinaclinicalisolateofitrichophytonrubrumi |