Mollicutes (“mycoplasmas”) are host-restricted prokaryotes and the simplest self-replicating organisms evolved from Gram-positive ancestors, which are characterized by a low GC content, small genomes (0.6–1.35 Mb), no cell wall, reduced coding capacity, and limited metabolic capacities (Sirand-Pugnet et al., 2007). Despite undergoing reductive evolution, mycoplasmas primarily colonize the mucosa of the respiratory and urogenital tracts, as well as the joints of vertebrate hosts, including multiple livestock, wild animal species, and humans. Some mycoplasmas are successful pathogens capable of establishing infection, which can result in significant socioeconomic consequences (Razin et al., 1998; Rosengarten et al., 2001; Citti and Blanchard, 2013; Arfi et al., 2021). Intrinsic antibiotic resistance, rapid tolerance to chemotherapeutic agents, and co-infection with other pathogenic species are causing growing concerns of mycoplasmas in both the medical and veterinary fields (Gautier-Bouchardon, 2018; Chen et al., 2023).

The growing body of sequence data has improved understanding of the structure and dynamics of mycoplasmas. However, genomic studies have revealed that mycoplasmas lack the classical repertoire of virulence genes common to pathogenic species. The molecular mechanisms underlying the pathogenesis of mycoplasmas in host calls include adhesion to the host respiratory epithelium, cell damage caused by cytotoxic metabolic compounds and the releasome, and modulation of the host microbicidal response. Experimentally confirmed virulence factors of mycoplasmas include the community-acquired respiratory distress syndrome toxin, hydrogen peroxide, and hydrogen sulfide (Burki et al., 2015; Benedetti et al., 2020; Leal Zimmer et al., 2020; Gaurivaud and Tardy, 2022). Limited information about the classical repertoire of virulence genes has impeded identification of the virulence genes and elucidation of the pathogenesis of mycoplasmas. In addition, the lack of appropriate genetic tools for genome manipulation has limited research on the virulence factors of mycoplasmas. Transposon-based vectors have been successfully applied for random insertion and inactivation of target genes of the mycoplasma genome (Dybvig et al., 2000; Kenri et al., 2004; Baranowski et al., 2010; Rideau et al., 2019). Over the past decade, synthetic biology has been successfully applied to several phylogenetically related species, but yet limited with other species (Gibson et al., 2008a; Schieck et al., 2016). Recently, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been successfully applied to silence target genes in the mycoplasma genome (Mariscal et al., 2018; Evsyutina et al., 2022). However, a more accurate, stable, and efficient genetic editing tool is urgently needed.

Natural plasmids of mycoplasmas are transmitted between species sharing a common host (Breton et al., 2012). OriC plasmids were developed to examine gene function in mycoplasmas, mesoplasmas, and spiroplasmas (Halbedel and Stulke, 2007; Maglennon et al., 2013; Renaudin et al., 2015; Matteau et al., 2017). In addition to these plasmids, this review summarizes current genetic tools to manipulate the mycoplasma genome. Current applications are classified as homologous recombination (HR), transposons, CRISPR/Cas systems, and synthetic biology. In addition, the development and optimization of CRISPR/Cas systems as novel genome editing tools for mycoplasmas are discussed.

HR is essential to access redundant genetic information encoded by sister chromatids or homologous chromosomes when both strands of the DNA double helix are compromised to support DNA replication and repair double-strand breaks (DSBs) (Wright et al., 2018). The mechanism of HR to repair DSBs has been widely applied for editing of bacterial genomes, but relatively few studies have investigated HR in mycoplasmas. Nonetheless, HR has been successfully used to edit the genomes of various Mycoplasma species, including M. gallisepticum (Cao et al., 1994; Lee et al., 2008), M. genitalium (Dhandayuthapani et al., 1999, 2001), M. capricolum subsp. capricolum (M. capricolum) (Janis et al., 2005), M. pneumoniae (Krishnakumar et al., 2010), and M. hyopneumoniae (Clampitt et al., 2021). HR was also used to integrate homologous DNA through a plasmid into the mycoplasma genome. However, success of this process was very low because of the lack of an efficient recombinase and transformation procedures. Therefore, an exogenous recombinase was applied to edit the genome of Mycoplasma gallisepticum to overcome the low efficiency of HR. The recE and recT genes of Bacillus subtilis were cloned into transposon-based vectors and integrated into the genome after transformation. The recombination templates in oriC plasmids were transformed into M. gallisepticum strains expressing RecE and RecT to generate a RecET-like system for precise recombination events leading to short deletions, the addition of resistance genes, and replacement of short genome regions (Ipoutcha et al., 2022). An oligonucleotide “recombineering” method for M. pneumoniae was also developed with the GP35 recombinase of B. subtilis to generate point mutations or deletion of larger fragments. Then, the CRISPR/Cas9 system was used to counter-select non-edited cells (Pinero-Lambea et al., 2020). In addition, the recombinase RecA of Escherichia coli was shown to enhance targeted HR in Mycoplasma mycoides subsp. capri and M. hyorhinis (Allam et al., 2010; Ishag et al., 2017).

Transposons are mobile genetic elements that evolved to execute highly efficient integration of genes into the host genome. As the most prominent mechanism, a transposase mediates excision of an element from the donor location and facilitates integration into a different locus of the genome (Sandoval-Villegas et al., 2021). Transposons are also widely used to integrate genes into the host genome. The transposons Tn916 and Tn4001, in addition to related derivatives, have been successfully used in Mollicutes (Halbedel and Stulke, 2007). Transposon Tn916, which was originally isolated from Enterococcus faecalis, is a conjugative 18-kb transposable element containing the xis-Tn/int-Tn genes for excision/integration, the tetM tetracycline resistance genes, a set of genes for intercellular transfer, and two imperfect inverted repeat sequence (20–60 bp) at both ends (Franke and Clewell, 1981; Clewell et al., 1988, 1995). Transposon Tn4001, originally isolated from Staphylococcus aureus, is a 4.5-kb composite containing an IS256 sequence at both ends of the aac-aphD gene, which confers resistance to gentamicin, kanamycin, and tobramycin (Lyon et al., 1984).

Integration of a transposon into the mycoplasma and acholeplasma genomes was first reported in Mycoplasma pulmonis and Acholeplasma laidlawii, respectively (Dybvig and Cassell, 1987). However, integration of Tn916 occurs at preferred hot spots and, thus, is less suitable for saturating transposon mutagenesis (Nelson et al., 1997). In 1989, Tn4001 was first integrated into the M. pulmonis genome, although the transposase was deleted to prevent reintegration and loss of the transposon by re-excision. The derivative Tn4001 is also called mini-Tn4001 (Zimmerman and Herrmann, 2005). To date, transposons have been widely used to construct mutant libraries of several mycoplasmas. For instance, Tn4001 and mini-Tn4001 were used to construct mutant libraries of M. pneumoniae to screen for essential genes and those associated with gliding motility (Hasselbring et al., 2006; Lluch-Senar et al., 2015). Tn916 and Tn4001 were also used to construct two mutant libraries of M. gallisepticum to screen for genes that regulate biofilm formation (Whetzel et al., 2003; Wang et al., 2017). In addition, mini-Tn4001 was applied to construct mutant libraries of M. agalactiae and M. bovis to identify genes that regulate nutrient acquisition from cells, genes that affect colonization and diffusion in the host cell, as well as other essential genes, such as those that code for adhesin proteins (Baranowski et al., 2010, 2014; Hegde et al., 2016; Josi et al., 2019; Zhu et al., 2020). Furthermore, mini-Tn4001, Tn4001, and Tn916 were successfully used to construct mutant libraries to identify the essential genes of various species of mycoplasmas, including M. genitalium, M. bovis, M. pulmonis, M. hyopneumoniae, M. mycoides, M. hominis, and M. hyorhinis, in addition to Ureaplasma parvum (Hutchison et al., 1999; Glass et al., 2006; French et al., 2008; Maglennon et al., 2013; Aboklaish et al., 2014; Sharma et al., 2014; Hutchison et al., 2016; Rideau et al., 2019; Trueeb et al., 2019). Also, mini-Tn4001 was used to express mCherry, Mko2, and mNeonGreen in M. bovis and M. mycoides subsp. mycoides to investigate host-pathogen interactions (Bonnefois et al., 2016). Tn4001 was also applied to examine the localization of green fluorescent protein (GFP)-tagged proteins of M. pneumoniae in mycoplasma cells (Kenri et al., 2004).

The transformation efficiency of Tn4001 is reported to significantly vary among different species, resulting in differences in transposition efficiency. The EF-Tu regulatory region of mycoplasma species was cloned into a transposon-based vector to regulate transposase expression and the antibiotic resistance marker to overcome this disadvantage. This derivative transposon, named SynMyco, was shown to increase transformation efficiency in M. gallisepticum, M. feriruminatoris, and M. agalactiae, but not M. pneumoniae (Montero-Blay et al., 2019). Recently, a LoxTnSeq system was developed to delete a large random genome fragment. Sequences of loxP were inserted into the genome of M. pneumoniae combined with expression of the exogenous recombinase Cre, while the large DNA fragment with a loxP sequence at both ends was deleted (Shaw et al., 2020). In addition, Tn5 was applied to generate mutant libraries of M. mycoides, Spiroplasma citri, and Mesoplasma florum (Mutaqin et al., 2011; Hutchison et al., 2016; Baby et al., 2018).

CRISPR-based genetic tools have revolutionized the field of genome engineering in eukaryotes and prokaryotes since first introduced in 2012. The classical CRISPR/Cas9 system uses a single-guide RNA (sgRNA) to target Cas9 nuclease to the desired DNA locus and create a site-specific DSB to the DNA. Genome editing is dependent on the repair machinery of the cell, including non-homologous end joining and homology-directed repair of DSBs (Jinek et al., 2012). Both endogenous and exogenous CRISPR/Cas systems have been applied to edit mycoplasma genomes (Table 1). In addition, the exogenous CRISPR/Cas system has been used to kill mycoplasma. Due to the lack of efficient non-homologous end joining and homology-directed repair pathways to repair DSBs, the CRISPR/Cas9 system was applied to break the genome of M. pneumoniae and limit growth (Broto et al., 2022). This system was also used to counter-select non-edited mycoplasma cells and to recover edited mycoplasma clones with limited screening of surviving cells (Pinero-Lambea et al., 2020). Moreover, an endogenous CRISPR/Cas system was used to edit the ksgA and munA genes of M. gallisepticum. The endogenous Cas protein of M. gallisepticum, which is reported to cut DNA, shares 26% amino acid sequence similarity with the type II-A Cas9 of S. aureus (Mahdizadeh et al., 2020; Klose et al., 2022). Bioinformatics revealed that the genome carried a gene with significant homology with the Ku and LigD genes of B. subtilis, which are the key elements of the non-homologous end joining repair system. These findings demonstrate that DNA repair systems differ among mycoplasma species. Further studies of DNA repair systems will contribute to further applications of the CRISPR/Cas system for genome editing of mycoplasmas.

Considering the low efficiency of repairing DSBs, endonuclease-deficient Cas9 (dCas9)-based gene editing tools, including CRISPR interference (CRISPRi), DNA base editing, and inducible CRISPRi, were applied to edit the genomes of mycoplasmas. Cas9 base-editing systems have been constructed to multiply the genome without inducing DSBs. DNA base editing systems combine a catalytically inactive form of Cas9 fused with a cytosine deaminase (CBE). This Cas9/CBE fusion protein is guided to specific loci by sgRNAs, where CBE catalyzes deamination of cytosine into uracil, which is recognized as thymine after replication. The C:G to A:T transition allows for insertion of a stop codon into the genome of M. bovis, M. mycoides subsp. mycoides, and M. gallisepticum. In addition, whole-genome sequencing revealed that this genome editing system is efficient with limited induction of off-target mutations (Lpoutcha et al., 2022). For the CRISPRi system, the dCas9 is guided to the loci by sgRNA and inhibits expression of the target gene by interfering with transcriptional elongation by binding of RNA polymerase or associated transcription factors (Qi et al., 2013). A single-plasmid transposon-based CRISPRi system was applied for genome editing of M. gallisepticum and M. hominis (Evsyutina et al., 2022). In this paper, the inactive Cas9 of Streptococcus pyogenes was cloned into the transposon-based vector pRLM5L2 and expressed in mycoplasma cells. However, expression of dCas9 had no significant effect on the growth rate of mycoplasma cells (Evsyutina et al., 2022). Moreover, an inducible CRISPRi system was also developed for M. pneumoniae and M. mycoides. With this system, the tetracycline operator regulates expression of the exogenous inactive form of Cas9, whereas tetracycline is used to induce expression of dCas9 and inhibit expression of the target gene (Mariscal et al., 2018). However, the effect of exogenous Cas9/dCas9 on the growth of mycoplasma when used to edit the genome remains unclear.

Synthetic biology strategies, which use yeast cells to engineer and transfer a bacterial genome into a recipient cell, have been applied to edit the genomes of M. genitalium, M. mycoides subsp. capri, and M. mycoides (Table 2), although gene editing is dependent on genome synthesis (Gibson et al., 2008a,b; Lartigue et al., 2009; Gibson et al., 2010). As compared to a synthetic genome, the genomes of M. genitalium, M. pneumoniae, M. mycoides subsp. capri, M. hominis, Mesoplasma florum, and A. laidlawii were cloned into yeast cells as circular centromeric plasmids (Karas et al., 2012; Rideau et al., 2017; Baby et al., 2018). Then, the restriction endonuclease AscI was used to create DSBs in the circular plasmids and a DNA fragment was inserted using HR in yeast cells. The mycoplasma genome appeared stable in yeast cells and provided a platform to engineer the mycoplasma genome in vivo (Benders et al., 2010). Besides cloning of the mycoplasma genome in yeast cells, the whole genome integrated with a yeast vector was transformed (Lartigue et al., 2009). Based on this platform, TREC (tandem repeat coupled with endonuclease cleavage), RMCE (Cre/loxP-based recombinase-mediated cassette exchange), TREC-IN (TREC-assisted gene knock-in), CreasPy-Cloning, Marker-less/driven, and meiotic recombination methods were developed to edit the genome of mycoplasmas in yeast cells. The TREC method uses a DNA cassette containing a knock-out CORE consisting of an 18-bp I-SceI recognition site, the SCEI gene under the control of the GAL1 promoter, and the URA3 marker. A DNA fragment homologous to the sequence upstream of the target site was used to insert into the genome of M. genitalium by HR in yeast cells, which generated tandem repeat sequences flanking the knock-out CORE. The inducible expression of I-SceI generated DSBs and promoted intra-molecular HR between the repeat sequences for excision of the CORE (Noskov et al., 2010; Schieck et al., 2016). The RMCE method uses a DNA cassette containing the cre gene under the control of the GAL1 promoter and URA3 marker flanked by the loxP sequence, where inducible expression of the Cre recombinase was used to insert DNA fragments into the mycoplasma genome (Noskov et al., 2010, 2015). The TREC-IN method was developed based on the TREC method and used to insert the target gene into the mycoplasma genome, where the gene was located at the 3′ end of the knock-out CORE and remained in the genome after excision of the CORE (Chandran et al., 2014; Lartigue et al., 2019). The CreasPy-Cloning method is a recently developed approach for simultaneous cloning and engineering of mycoplasma genomes in yeast cells. This method combines the abilities of Cas9 to cleave DNA at a specific locus and the efficient HR of yeast cells to edit the mycoplasma genome, which was transformed into yeast cells in whole (Kannan et al., 2016; Tsarmpopoulos et al., 2016; Rideau et al., 2017; Ruiz et al., 2019; Talenton et al., 2022). The Marker-less/driven method with EZ-Tn5™ transposase was used to successfully insert DNA fragments into the genome of M. mycoides in vitro and then the edited genome was transformed into the mycoplasma (Karas et al., 2014). The meiotic recombination method replaces the individual gene with the GFP marker in the mycoplasma genome by HR in yeast cells. Then, the yeast cells with GFP markers at different loci are mixed and the progressively clustering genomic segments are deleted by meiotic recombination between the mycoplasma genomes harbored in yeast cells (Sugiyama et al., 2005; Suzuki et al., 2011, 2015).

Each of the above methods requires transfer of the edited genome into the recipient cell to generate mutants. Such transfer is possible for several species related to the M. mycoides cluster, but not members of other phylogenetic groups. The recombinase-assisted genomic engineering (RAGE) method was developed to overcome this disadvantage with genome editing of M. pneumoniae. This method uses the transformation-associated recombination mechanism, where a DNA fragment of the mycoplasma genome, the cre gene, the selection marker aac-aph, and a linearized bacterial artificial chromosome-yeast artificial chromosome shuttle vector are assembled in a recombinant plasmid in yeast cells. Then, the plasmid is extracted and transformed into E. coli cells for amplification. Thereafter, M. pneumoniae cells are transformed with numerous plasmids and the genome is edited by RMCE. Based on this new strategy, the TREC, Cre/loxP, TREC-IN, and CreasPy-Cloning methods could be used to edit fragments of mycoplasma genomes in yeast cells (Garcia-Morales et al., 2020).

Lastly, the “targeting-induced local lesions in genomes” method is a reverse-genetic method to edit the M. hominis genome, which combines point mutations (C-G to T-A) induced by ethyl methane sulfonate with a DNA screening technique to generate a library of M. hominis mutants (Pereyre et al., 2018).

Due to the lack of efficient genetic tools, it is challenging to elucidate the functional genomics of mycoplasmas. Although several methods have been developed to knock-out or knock-down target genes in the genome (Figure 1; Table 3), stable, efficient, and universal genetic tools have not yet been established for mycoplasmas.

Transposons are conventionally used to generate mutant libraries, which can be combined with smart screening systems to identify genes that regulate nutrient acquisition from host cells, affect colonization and diffusion in the host cell, as well as other essential genes, such as those that code for adhesin proteins. However, the lack of genetic tools for site-directed mutagenesis in many mycoplasma species has impeded further clarification of the interactions between mycoplasmas and host cells. The CRISPR/Cas system and synthetic biology were recently applied to knock-out target genes. These studies provide new insights into the genetic manipulation of mycoplasmas. Notably, the CRISPRi system is more appropriate than knock-down/out methods to analyze the functions of essential genes. In addition, gene function can be confirmed by trans-complementation of mutants based on oriC plasmids. This review summarizes the results of recent studies and discusses strategies for the development of accurate and efficient tools for editing of mycoplasma genomes.

GZ and DL write original manuscript. ML and YW designed and revised the article. All authors contributed to the article and approved the submitted version.

This work was supported by the Youth Program of National Natural Science Foundation of China (#32102672), National Natural Science Foundation of China Joint Fund Project (U22A20505).

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.

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