A Robust CRISPR Interference Gene Repression System in Pseudomonas
Name
JB00575-17R1_Merged_PDF.pdf
Description
Accepted version
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1.03 MB
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Checksum (MD5)
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Author(s)
Tan, Sue Zanne
Reisch, Christopher R.
Prather, Kristala L. J.
Alternative Title
A Robust CRISPR Interference Gene Repression System in Pseudomonas
Date Issued
March 2018
Journal
Journal of Bacteriology
Publisher
American Society for Microbiology
Version
Author's final manuscript
Abstract
© 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.
MIT Department
Massachusetts Institute of Technology. Department of Chemical Engineering
Terms of Use
Creative Commons Attribution-Noncommercial-Share Alike
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DOI of Published Version
https://doi.org/10.1128/jb.00575-17
