Genome Editing of Veterinary Relevant Mycoplasmas Using a CRISPR-Cas Base Editor System

Abstract

Mycoplasmas are minimal bacteria that infect humans, wildlife, and most economically relevant livestock species. Mycoplasma infections cause a large range of chronic inflammatory diseases, eventually leading to death in some animals. Due to the lack of efficient recombination and genome engineering tools for most species, the production of mutant strains for the identification of virulence factors and the development of improved vaccine strains is limited. Here, we demonstrate the adaptation of an efficient Cas9-Base Editor system to introduce targeted mutations into three major pathogenic species that span the phylogenetic diversity of these bacteria: the avian pathogen Mycoplasma gallisepticum and the two most important bovine mycoplasmas, Mycoplasma bovis and Mycoplasma mycoides subsp. mycoides. As a proof of concept, we successfully used an inducible SpdCas9-pmcDA1 cytosine deaminase system to disrupt several major virulence factors in these pathogens. Various induction times and inducer concentrations were evaluated to optimize editing efficiency. The optimized system was powerful enough to disrupt 54 of 55 insertion sequence transposases in a single experiment. Whole-genome sequencing of the edited strains showed that off-target mutations were limited, suggesting that most variations detected in the edited genomes are Cas9-independent. This effective, rapid, and easy-to-use genetic tool opens a new avenue for the study of these important animal pathogens and likely the entire class Mollicutes. IMPORTANCE Mycoplasmas are minimal pathogenic bacteria that infect a wide range of hosts, including humans, livestock, and wild animals. Major pathogenic species cause acute to chronic infections involving still poorly characterized virulence factors. The lack of precise genome editing tools has hampered functional studies of many species, leaving multiple questions about the molecular basis of their pathogenicity unanswered. Here, we demonstrate the adaptation of a CRISPR-derived base editor for three major pathogenic species: Mycoplasma gallisepticum, Mycoplasma bovis, and Mycoplasma mycoides subsp. mycoides. Several virulence factors were successfully targeted, and we were able to edit up to 54 target sites in a single step. The availability of this efficient and easy-to-use genetic tool will greatly facilitate functional studies of these economically important bacteria.

Keywords: CRISPR-Cas9; animal pathogens; genome editing; minimal cell; mycoplasma.

FIG 1

Targeting of the ksgA gene in M. gallisepticum using the pmcDA1 deaminase-encoding plasmid. (A) Scheme of the CBE expression cassette. The synthetic cassette is composed of (i) a sgRNA (red), including the 20-nucleotide target spacer (dark blue) under the control of the spiralin promoter (brown), and (ii) a codon-optimized hybrid protein composed of S. pyogenes inactivated Cas9 (SpdCas9, green), linkers (purple), the pmcDA1 deaminase protein (light blue), and uracil glycosylase inhibitor (UGI) (dark purple) under the control of the Pxyl/tetO2 inducible promoter (dark green). Fibril terminators from S. citri (light green) were added downstream of the sgRNA and hybrid protein expression cassettes. The sequence of the ksgA target is represented here. Cytosine residues that are susceptible to deamination are colored in red. The positions of bases in the target are indicated below each base, with numbering starting at the PAM sequence. (B). The percentage of bases found in the population of transformants was determined by Sanger sequencing of the complementary strand. The chromatograms were analyzed using EditR software and are represented in a table for each nucleotide position in the target sequence. Positions 16 and 17 are framed in a dotted rectangle. (C) Schematic diagram of the base-editing experiment in M. gallisepticum (Mgal) and an indication of various checkpoints (Sanger results and EditR analysis) for screening until isolated mutants are obtained.

FIG 2

Targeting of three virulence factors of M. gallisepticum to explore the editing window of the CBE system in M. gallisepticum. The 20-nucleotide target sites were sequenced and are represented here for the crmA (A), gapA (B), and cysP (C) genes before and after the base-editing experiments. Cytosines susceptible to deamination (or guanine in the reverse strand) are framed in red, and those that were deaminated are framed in green. The percentage of each base is shown in the tables, as determined using the Sanger sequencing.ab file and EditR software. Edited bases are highlighted in green. For the crmA target, the complementary strand was sequenced. (D) The 20-nucleotide target sites are shown for the three targets. The position of each base in the target is indicated below each nucleotide and corresponds to the nucleotide position in the target upstream of the PAM sequence. Red letters represent undeaminated cytosines, and green letters represent converted thymines. A scheme based on the three experiments and highlighting the editing window is shown at the bottom. As shown in the scheme, cytosines located at positions 16 to 20 can be CBE-targeted.

FIG 3

Disruption of the MnuA nuclease-encoding gene by base editing in M. bovis. A schematic diagram based on a base-editing experiment in M. bovis. After transformation of M. bovis (Mbov) with the plasmid pMT85_SpdCas9_pmcDA1, cells were propagated in liquid media supplemented with gentamicin. After three passages in liquid medium (P1 to P3), an inducer was added to the cell suspension. Aliquots were collected at each passage and after induction to monitor the target site sequence by Sanger sequencing and EditR analysis. Sequencing chromatograms of the complementary strand are presented along with the percentage of the base for each position of the target sequence. As shown in the diagram, cytosines susceptible to deamination are framed in red, and the edited bases are framed in green.

FIG 4

Multitargeting of IS1634 copies in the Mmm genome. (A) Schematic diagram of the Mmm T1/44 genome with 58 complete or truncated copies of the IS1634 transposases represented as solid lines. In the right panel, the targeted site within the IS1634 transposase gene is represented as a red bar. (B) Schematic diagram of the three induction steps of the mycoplasma CBE system for targeting 55 IS1634 sites using a single gRNA. The nucleotide sequence of the target is represented with the PAM sequence and framed by a red rectangle. Cytosines susceptible to deamination are shown in red. Fresh cultures of Mmm cells harboring the pMYCO1_SpdCas_pmcDA1 plasmid are represented as red-colored tubes. Cultures in exponential and stationary growth phases are indicated by orange-and yellow-colored tubes, respectively. Three consecutive culture steps were performed (including the starting culture) by a 1/100 dilution in fresh medium. At each step, induction was performed by adding aTC and incubating the cells until stationary-phase was reached. Deamination of target cytosines was evaluated by PCR and Sanger sequencing. The percentage of each base at each position was estimated from chromatograms using EditR software. The two positions of interest are highlighted in a dashed red rectangle. (C) Sanger sequencing results and base distribution in a selected isolated clone. Fully mutated positions are framed in green in the table and shown in green in the target sequence.