A Robust CRISPR Interference Gene Repression System in Pseudomonas
© 2018 American Society for Microbiology. Pseudomonas spp. are widely used model organisms in different areas of research. Despite the relevance of Pseudomonas in many applications, the use of protein depletion tools in this host remains limited. Here, we developed the CRISPR interference system for...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2021
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| Online Access: | https://hdl.handle.net/1721.1/134186 |
| _version_ | 1826196575082250240 |
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| author | Tan, Sue Zanne Reisch, Christopher R Prather, Kristala LJ |
| author_facet | Tan, Sue Zanne Reisch, Christopher R Prather, Kristala LJ |
| author_sort | Tan, Sue Zanne |
| collection | MIT |
| description | © 2018 American Society for Microbiology. Pseudomonas spp. are widely used model organisms in different areas of research. Despite the relevance of Pseudomonas in many applications, the use of protein depletion tools in this host remains limited. Here, we developed the CRISPR interference system for gene repression in Pseudomonas spp. using a nuclease-null Streptococcus pasteurianus Cas9 variant (dead Cas9, or dCas9). We demonstrate a robust and titratable gene depletion system with up to 100-fold repression in β-galactosidase activity in P. aeruginosa and 300-fold repression in pyoverdine production in Pseudomonas putida. This inducible system enables the study of essential genes, as shown by ftsZ depletions in P. aeruginosa, P. putida, and Pseudomonas fluorescens that led to phenotypic changes consistent with depletion of the targeted gene. Additionally, we performed the first in vivo characterization of protospacer adjacent motif (PAM) site preferences of S. pasteurianus dCas9 and identified NNGCGA as a functional PAM site that resulted in repression efficiencies comparable to the consensus NNGTGA sequence. This discovery significantly expands the potential genomic targets of S. pasteurianus dCas9, especially in GC-rich organisms. |
| first_indexed | 2024-09-23T10:29:53Z |
| format | Article |
| id | mit-1721.1/134186 |
| institution | Massachusetts Institute of Technology |
| language | English |
| last_indexed | 2024-09-23T10:29:53Z |
| publishDate | 2021 |
| publisher | American Society for Microbiology |
| record_format | dspace |
| spelling | mit-1721.1/1341862022-04-01T16:26:07Z A Robust CRISPR Interference Gene Repression System in Pseudomonas A Robust CRISPR Interference Gene Repression System in Pseudomonas Tan, Sue Zanne Reisch, Christopher R Prather, Kristala LJ © 2018 American Society for Microbiology. Pseudomonas spp. are widely used model organisms in different areas of research. Despite the relevance of Pseudomonas in many applications, the use of protein depletion tools in this host remains limited. Here, we developed the CRISPR interference system for gene repression in Pseudomonas spp. using a nuclease-null Streptococcus pasteurianus Cas9 variant (dead Cas9, or dCas9). We demonstrate a robust and titratable gene depletion system with up to 100-fold repression in β-galactosidase activity in P. aeruginosa and 300-fold repression in pyoverdine production in Pseudomonas putida. This inducible system enables the study of essential genes, as shown by ftsZ depletions in P. aeruginosa, P. putida, and Pseudomonas fluorescens that led to phenotypic changes consistent with depletion of the targeted gene. Additionally, we performed the first in vivo characterization of protospacer adjacent motif (PAM) site preferences of S. pasteurianus dCas9 and identified NNGCGA as a functional PAM site that resulted in repression efficiencies comparable to the consensus NNGTGA sequence. This discovery significantly expands the potential genomic targets of S. pasteurianus dCas9, especially in GC-rich organisms. 2021-10-27T19:58:33Z 2021-10-27T19:58:33Z 2018 2019-07-22T13:59:42Z Article http://purl.org/eprint/type/JournalArticle https://hdl.handle.net/1721.1/134186 en 10.1128/JB.00575-17 Journal of Bacteriology Creative Commons Attribution-Noncommercial-Share Alike http://creativecommons.org/licenses/by-nc-sa/4.0/ application/pdf American Society for Microbiology Prof. Prather |
| spellingShingle | Tan, Sue Zanne Reisch, Christopher R Prather, Kristala LJ A Robust CRISPR Interference Gene Repression System in Pseudomonas |
| title | A Robust CRISPR Interference Gene Repression System in Pseudomonas |
| title_full | A Robust CRISPR Interference Gene Repression System in Pseudomonas |
| title_fullStr | A Robust CRISPR Interference Gene Repression System in Pseudomonas |
| title_full_unstemmed | A Robust CRISPR Interference Gene Repression System in Pseudomonas |
| title_short | A Robust CRISPR Interference Gene Repression System in Pseudomonas |
| title_sort | robust crispr interference gene repression system in pseudomonas |
| url | https://hdl.handle.net/1721.1/134186 |
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