Abstract
Mycoplasmas are pathogenic, genome-reduced bacteria. The development of such fields of science as system and synthetic biology is closely associated with them. Despite intensive research of different representatives of this genus, genetic manipulations remain challenging in mycoplasmas. Here we demonstrate a single-plasmid transposon-based CRISPRi system for the repression of gene expression in mycoplasmas. We show that selected expression determinants provide a level of dCas9 that does not lead to a significant slow-down of mycoplasma growth. For the first time we describe the proteomic response of genome-reduced bacteria to the expression of exogenous dcas9. The functionality of the resulting vector is confirmed by targeting the three genes coding transcription factors-fur, essential spxA, whiA, and histone-like protein hup1 in Mycoplasma gallisepticum. As a result, the expression level of each gene was decreased tenfold and influenced the mRNA level of predicted targets of transcription factors. To illustrate the versatility of this vector, we performed a knockdown of metabolic genes in a representative member of another cluster of the Mycoplasma genus-Mycoplasma hominis. The developed CRISPRi system is a powerful tool to discover the functioning of genes that are essential, decipher regulatory networks and that can help to identify novel drug targets to control Mycoplasma infections.