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

Ipoutcha, Thomas; Rideau, Fabien; Gourgues, Géraldine; Arfi, Yonathan; Lartigue, Carole; Blanchard, Alain; Sirand‐Pugnet, Pascal

doi:10.1128/aem.00996-22

Cited by 13 publications

(32 citation statements)

References 107 publications

(133 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The gene encoding MnuA in M. bovis is MBOVPG45_0215 and its homolog in M. agalactiae is MAG_5900. While the phenotype of the M. bovis mnuA mutant was described in Ipoutcha et al, 2022 48 , the phenotype of the M. agalactiae mnuA mutant is presented here (Figure S5).…”

Section: Assessment Of the Creaspy-fusion's Limitations Using Mycopla...mentioning

confidence: 81%

“…The gene encoding MnuA in M. bovis is MBOVPG45_0215 and its homolog in M. agalactiae is MAG_5900. While the phenotype of the M. bovis mnuA mutant was described in Ipoutcha et al ., 2022 48 , the phenotype of the M. agalactiae mnuA mutant is presented here ( Figure S5 ). Just like M. bovis (and M. gallisepticum above), M. agalactiae mnuA inactivation resulted in a significant reduction of DNA hydrolysis when mutant cells were incubated with DNA substrates, either linear or circular.…”

Section: Resultsmentioning

confidence: 99%

“…In this mutant, a frameshift at the beginning of the cas9 nucleotide sequence (position 51) leads to premature termination of the protein expression and loss of function (Thomas Ipoutcha, personal communication). The two other mutants (an mnuA mutant and a cas9-mnuA double mutant) (Figure 3A) were generated for this study using the CRISPR-Cas9 base editor system (CBE), which was recently adapted to mycoplasmas 48 . We chose not to include a "GCW_RS00180 mutant" initially because a study performed on M. bovis PG45 T had shown that among all nuclease coding genes in this species (3 in total), mnuA was responsible for the majority of the nuclease activity detectable in vitro 43 .…”

Section: Overcoming a Potential Barrier For M Gallisepticum Genome Cl...mentioning

confidence: 99%

“…With this goal, we chose M. bovis PG45 T and M. agalactiae PG2 T , two other mycoplasmas pathogenic for ruminants and that are phylogenetically close to each other. Taking into account the results obtained above, mnuA mutants of these mycoplasmas were produced by targeted base editing using Cas9 deaminase 48 . The gene encoding MnuA in M. bovis is MBOVPG45_0215 and its homolog in M. agalactiae is MAG_5900.…”

Section: Assessment Of the Creaspy-fusion's Limitations Using Mycopla...mentioning

confidence: 99%

“…M. gallisepticum and M. agalactiae were transformed with the pTi4.0_Sp_pmcda_NucGalli0070 or pFRIT4.0-mnuA plasmids using protocols described by Ipoutcha et al, 2022 48,58 and Zhu et al, 2020 59 respectively. Transformants were selected on appropriate solid media containing antibiotics (10µg.ml -1 of puromycin for M. gallisepticum and with 100 µg.mL -1 of gentamycin for M. agalactiae).…”

Section: Construction Of Grna Plasmids For Simple Target Deletionmentioning

confidence: 99%

See 4 more Smart Citations

Combining fusion of cells with CRISPR-Cas9 editing for the cloning of large DNA fragments or complete bacterial genomes in yeast

Guesdon

Gourgues

Rideau

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

The genetic engineering of genome fragments larger than 100 kbp is challenging and requires both specific methods and cloning hosts. The yeast Saccharomyces cerevisiae is considered as a host of choice for cloning and engineering whole or partial genomes from viruses, bacteria, and algae. Several methods are now available to perform these manipulations, each with its own limitations. In order to extend the range of in-yeast cloning strategies, a new approach combining two already described methods, the Fusion cloning and the CReasPy-Cloning, was developed. The CReasPy-Fusion method allows the simultaneous cloning and engineering of megabase-sized genomes in yeast by fusion of bacterial cells with yeast spheroplasts carrying the CRISPR-Cas9 system. With this new approach, we demonstrate the feasibility of cloning and editing whole genomes from several Mycoplasma species belonging to different phylogenetic groups. We also show that CReasPy-Fusion allows the capture of large genome fragments with high efficacy, resulting in the successful cloning of selected loci in yeast. We finally identify bacterial nuclease encoding genes as barriers for CReasPy-Fusion by showing that their removal from the donor genome improves cloning efficacy.

show abstract

Section: Assessment Of the Creaspy-fusion's Limitations Using Mycopla...mentioning

confidence: 81%

Section: Resultsmentioning

confidence: 99%

Section: Overcoming a Potential Barrier For M Gallisepticum Genome Cl...mentioning

confidence: 99%

Section: Assessment Of the Creaspy-fusion's Limitations Using Mycopla...mentioning

confidence: 99%

Section: Construction Of Grna Plasmids For Simple Target Deletionmentioning

confidence: 99%

See 3 more Smart Citations

Combining fusion of cells with CRISPR-Cas9 editing for the cloning of large DNA fragments or complete bacterial genomes in yeast

Guesdon

Gourgues

Rideau

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

Contagious Bovine Pleuropneumonia

Thiaucourt¹,

Nwankpa²,

Amanfu³

2021

Veterinary Vaccines

View full text Add to dashboard Cite

The protection rate of T1/44 and T1sr vaccines assessed in two independent vaccine trials varied between 33 and 67% (Thiaucourt et al., 2000). Animals were challenged against local pathogenic strains, 3 months after single vaccination. Further experiments demonstrated that T1/44 confers longer immunity than T1sr after a single dose, while a booster vaccination received 12 months after initial immunisation increased the efficacy of both vaccines (> 80%; Wesongaet al., 2000). In order to be effective vaccines must contain at least 10 7 CFU/dose of live Mmm (WOAH, 2021).Both vaccines confer low efficacy at primo-vaccination and short-term immunity, requiring re-vaccinations every 6 and 12 months respectively for T1sr and T1/44. T1/44 has residual virulence, which results in occasional cases of severe inflammation at the site of injection called Willems' reactions. These local reactions can evolve into systemic disease and may even result in death of the animals in the absence of antibiotic treatments, which can discourage vaccine acceptance by farmers . Willems' reactions are observed mainly upon first vaccination and with low occurrence after re-vaccination (Muuka et al., 2014). No side effects have been reported for T1sr.Both T1/44 and T1sr are currently produced as freeze-dried live vaccines with cold chain requirements until delivery. After reconstitution, delivery time is restricted to 30 min -2 hrs. In field conditions and with hot temperatures, viability rapidly drops below the minimum recommended titre affecting vaccine efficacy.The quality of CBPP vaccines is known to differ among producers / batches, which may result in variable vaccine efficacy. Due to the potential side effects and cold chain requirements, vaccine delivery should be restricted to fully trained veterinary personnel. GAPS :Vaccine safety: Side effects associated with the T1/44 strain are perceived as an important issue. For example, T1/44 is not accepted in some countries due to reported adverse reactions and T1sr is used instead, although the frequency of vaccination campaigns is normally once per year. Vaccine delivery can exacerbate side effects, since applying inappropriate administration routes (intradermal or intramuscular instead of subcutaneous) or using inappropriate needle gauge, etc. can result in increased adverse reactions and, thus, reduced acceptability by farmers.Vaccine efficacy: The true efficacy of T1 vaccines in the field is not well known. Control programs based on vaccination alone are not sufficient for CBPP eradication in the absence of additional countermeasures such as animal movement control. For ex.: Northern Namibia and Western Zambia, where CBPP mass vaccination campaigns are delivered annually, are still struggling to control the disease, with many outbreaks declared in 2021 and 2022.Vaccine delivery: Fully trained personnel is needed to manage vaccination, since administration problems such as use of wrong diluents and vial sizes, inappropriate number of doses per vial, etc. can result in reduced viability a...

show abstract

Genome transplantation in Mollicutes

Labroussaa¹,

Torres-Puig²,

Jores³

2023

Genome Engineering

View full text Add to dashboard Cite

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

Abstract: 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.

Cited by 13 publications

References 107 publications

Combining fusion of cells with CRISPR-Cas9 editing for the cloning of large DNA fragments or complete bacterial genomes in yeast

Combining fusion of cells with CRISPR-Cas9 editing for the cloning of large DNA fragments or complete bacterial genomes in yeast

Contagious Bovine Pleuropneumonia

Genome transplantation in Mollicutes

Contact Info

Product

Resources

About