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Review
. 2023 May 18:14:1191812.
doi: 10.3389/fmicb.2023.1191812. eCollection 2023.

Gene editing tools for mycoplasmas: references and future directions for efficient genome manipulation

Gang Zhao  1   2   3 , Doukun Lu  3 , Min Li  1   2 , Yujiong Wang  1   2
Affiliations
Review

Gene editing tools for mycoplasmas: references and future directions for efficient genome manipulation

Gang Zhao et al. Front Microbiol. .

Abstract

Mycoplasmas are successful pathogens that cause debilitating diseases in humans and various animal hosts. Despite the exceptionally streamlined genomes, mycoplasmas have evolved specific mechanisms to access essential nutrients from host cells. The paucity of genetic tools to manipulate mycoplasma genomes has impeded studies of the virulence factors of pathogenic species and mechanisms to access nutrients. This review summarizes several strategies for editing of mycoplasma genomes, including homologous recombination, transposons, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system, and synthetic biology. In addition, the mechanisms and features of different tools are discussed to provide references and future directions for efficient manipulation of mycoplasma genomes.

Keywords: clustered regularly interspaced short palindromic repeats/Cas9 system; genome engineering; mycoplasma; synthetic biology; transposon.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Schematics of the genetic tools based on HR, transposons, the CRISPR/Cas9 system, and synthetic biology. HR: the heterologous recombinase (HeRec) and homologous recombinase (HoRec) were applied to edit the mycoplasma genome. Transposon: Tn4001, mini-Tn4001, Tn5, and Tn916 were used to insert transposons into the genome for gene knock-out. CRISPR/Cas9: the endogenous CRISPR/Cas system of M. gallisepticum and exogenous CRISPR/Cas9 system were used to edit the mycoplasma genome. The exogenous CRISPR/Cas9 system includes: (i) inactivated Cas9 fused with a CBE to induce C:G to T:A for insertion of a stop codon into the target gene; (ii) inactivated Cas9 for interfering with expression of the target gene; and (iii) activated Cas9 for knock-out of the target gene. Synthetic biology: the genome was edited by the TREC, CreasPy-Cloning, and RMEC methods in yeast cells and then transferred into recipient mycoplasma cells; the genome fragments of M. pneumoniae were edited by RAGE in E. coli cells and then transferred into M. pneumoniae recipient cells.
Figure 2
Figure 2
The ideas on improving the efficient of existing tools for editing of mycoplasma genomes. RM, restriction-modification; crRNA, CRISPR RNA.
See this image and copyright information in PMC

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