Tuning Gene Activity by Inducible and Targeted Regulation of Gene Expression in Minimal Bacterial Cells

Ana M. Mariscal, Shigeyuki Kakizawa, Jonathan Y. Hsu, Kazuki Tanaka, Luis González-González, Alicia Broto, Enrique Querol, Maria Lluch-Senar, Carlos Piñero-Lambea, Lijie Sun, Philip D. Weyman, Kim S. Wise, Chuck Merryman, Gavin Tse, Adam J. Moore, Clyde A. Hutchison, Hamilton O. Smith, Masaru Tomita, J. Craig Venter, John I. GlassJaume Piñol, Yo Suzuki

Research output: Contribution to journalArticlepeer-review

22 Citations (Scopus)


Functional genomics studies in minimal mycoplasma cells enable unobstructed access to some of the most fundamental processes in biology. Conventional transposon bombardment and gene knockout approaches often fail to reveal functions of genes that are essential for viability, where lethality precludes phenotypic characterization. Conditional inactivation of genes is effective for characterizing functions central to cell growth and division, but tools are limited for this purpose in mycoplasmas. Here we demonstrate systems for inducible repression of gene expression based on clustered regularly interspaced short palindromic repeats-mediated interference (CRISPRi) in Mycoplasma pneumoniae and synthetic Mycoplasma mycoides, two organisms with reduced genomes actively used in systems biology studies. In the synthetic cell, we also demonstrate inducible gene expression for the first time. Time-course data suggest rapid kinetics and reversible engagement of CRISPRi. Targeting of six selected endogenous genes with this system results in lowered transcript levels or reduced growth rates that agree with lack or shortage of data in previous transposon bombardment studies, and now produces actual cells to analyze. The ksgA gene encodes a methylase that modifies 16S rRNA, rendering it vulnerable to inhibition by the antibiotic kasugamycin. Targeting the ksgA gene with CRISPRi removes the lethal effect of kasugamycin and enables cell growth, thereby establishing specific and effective gene modulation with our system. The facile methods for conditional gene activation and inactivation in mycoplasmas open the door to systematic dissection of genetic programs at the core of cellular life.

Original languageEnglish
Pages (from-to)1538-1552
Number of pages15
JournalACS Synthetic Biology
Issue number6
Publication statusPublished - 2018 Jun 15


  • clustered regularly interspaced short palindromic repeats (CRISPR)
  • functional genomics
  • inducible promoters
  • mycoplasma
  • riboswitch
  • tetracycline-mediated repression

ASJC Scopus subject areas

  • Biomedical Engineering
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)


Dive into the research topics of 'Tuning Gene Activity by Inducible and Targeted Regulation of Gene Expression in Minimal Bacterial Cells'. Together they form a unique fingerprint.

Cite this