High-Level dCas9 Expression Induces Abnormal Cell Morphology in Escherichia coli

Along with functional advances in the use of CRISPR/Cas9 for genome editing, endonuclease-deficient Cas9 (dCas9) has provided a versatile molecular tool for exploring gene functions. In principle, differences in cell phenotypes that result from the RNA-guided modulation of transcription levels by dC...

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Published in:ACS synthetic biology 2018-04, Vol.7 (4), p.1085-1094
Main Authors: Cho, Suhyung, Choe, Donghui, Lee, Eunju, Kim, Sun Chang, Palsson, Bernhard, Cho, Byung-Kwan
Format: Article
Language:English
Subjects:
CRISPR-Associated Protein 9 - genetics
CRISPR-Associated Protein 9 - metabolism
Doxycycline - pharmacology
Escherichia coli - cytology
Escherichia coli - genetics
Escherichia coli - growth & development
Gene Editing - methods
Gene Expression Regulation, Bacterial
Genome, Bacterial
Microorganisms, Genetically-Modified - cytology
RNA, Messenger - genetics
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cited_by cdi_FETCH-LOGICAL-a339t-2bef5f9abbcaffefe7d8fc476bbfca52a389a55027df1cba48400c3706fa6eb53
cites cdi_FETCH-LOGICAL-a339t-2bef5f9abbcaffefe7d8fc476bbfca52a389a55027df1cba48400c3706fa6eb53
container_end_page 1094
container_issue 4
container_start_page 1085
container_title ACS synthetic biology
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creator Cho, Suhyung
Choe, Donghui
Lee, Eunju
Kim, Sun Chang
Palsson, Bernhard
Cho, Byung-Kwan
description Along with functional advances in the use of CRISPR/Cas9 for genome editing, endonuclease-deficient Cas9 (dCas9) has provided a versatile molecular tool for exploring gene functions. In principle, differences in cell phenotypes that result from the RNA-guided modulation of transcription levels by dCas9 are critical for inferring with gene function; however, the effect of intracellular dCas9 expression on bacterial morphology has not been systematically elucidated. Here, we observed unexpected morphological changes in Escherichia coli mediated by dCas9, which were then characterized using RNA sequencing (RNA-Seq) and chromatin immunoprecipitation sequencing (ChIP-Seq). Growth rates were severely decreased, to approximately 50% of those of wild type cells, depending on the expression levels of dCas9. Cell shape was changed to abnormal filamentous morphology, indicating that dCas9 affects bacterial cell division. RNA-Seq revealed that 574 genes were differentially transcribed in the presence of high expression levels of dCas9. Genes associated with cell division were upregulated, which was consistent with the observed atypical morphologies. In contrast, 221 genes were downregulated, and these mostly encoded proteins located in the cell membrane. Further, ChIP-Seq results showed that dCas9 directly binds upstream of 37 genes without single-guide RNA, including fimA, which encodes bacterial fimbriae. These results support the fact that dCas9 has critical effects on cell division as well as inner and outer membrane structure. Thus, to precisely understand gene functions using dCas9-driven transcriptional modulation, the regulation of intracellular levels of dCas9 is pivotal to avoid unexpected morphological changes in E. coli.
doi_str_mv 10.1021/acssynbio.7b00462
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Biol</addtitle><date>2018-04-20</date><risdate>2018</risdate><volume>7</volume><issue>4</issue><spage>1085</spage><epage>1094</epage><pages>1085-1094</pages><issn>2161-5063</issn><eissn>2161-5063</eissn><notes>ObjectType-Article-1</notes><notes>SourceType-Scholarly Journals-1</notes><notes>ObjectType-Feature-2</notes><notes>content type line 23</notes><abstract>Along with functional advances in the use of CRISPR/Cas9 for genome editing, endonuclease-deficient Cas9 (dCas9) has provided a versatile molecular tool for exploring gene functions. In principle, differences in cell phenotypes that result from the RNA-guided modulation of transcription levels by dCas9 are critical for inferring with gene function; however, the effect of intracellular dCas9 expression on bacterial morphology has not been systematically elucidated. 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source American Chemical Society:Jisc Collections:American Chemical Society Read & Publish Agreement 2022-2024 (Reading list)
subjects CRISPR-Associated Protein 9 - genetics
CRISPR-Associated Protein 9 - metabolism
Doxycycline - pharmacology
Escherichia coli - cytology
Escherichia coli - genetics
Escherichia coli - growth & development
Gene Editing - methods
Gene Expression Regulation, Bacterial
Genome, Bacterial
Microorganisms, Genetically-Modified - cytology
RNA, Messenger - genetics
title High-Level dCas9 Expression Induces Abnormal Cell Morphology in Escherichia coli
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