Journal: Journal of Bacteriology

Article Title: A targeted mutational strategy aiding in generating antisense RNA to knockdown the Ehrlichia chaffeensis P28-outer membrane protein 19 expression

doi: 10.1128/jb.00334-26

Figure Lengend Snippet: Confirmation of the mutant E. chaffeensis by PCR and whole-genome sequencing. ( A ) Schematic view of the duplex RNA strand formation of asRNA with mRNA of ECH_1143 . The entire predicted asRNA and sense RNA forming hybrids are presented in the image. ( B ) PCR analysis confirming the mutation generation. Primers annealing upstream (P1) and downstream (P5) of the mutation insertion region (genomic coordinates identified) were used to amplify DNA segments by PCR I, which were then resolved on an agarose gel; the expected larger 4.77 kb product was evident from the mutant genomic DNA (M) compared to the smaller 2.07 kb from wild-type genomic DNA (W). (L, 1 kb plus DNA ladder). Similarly, PCR II using primers P1 and P2 targeted to the insertion region and genomic region upstream of the insertion region yielded the expected amplicon of 2.18 kb only from the mutant. ( C ) Nanopore whole-genome sequencing reads were used to assemble the genome of the antisense mutant. The insertion segment was identified at the anticipated region within the E. chaffeensis genome. ( D ) Transcriptional analysis confirming the ECH_1143 asRNA. The presence of asRNA was assessed by RT-PCR using RNA isolated from wild-type (W) and mutant (M) E. chaffeensis in the macrophage cell line and tick cell line. RNA samples were reverse transcribed to generate cDNA using primer P4, which was designed to anneal downstream of the asRNA sequence, which was expected to be present only in the asRNA mutant. The cDNA was used as the template for PCR amplification with primers P3 and P4 yielding the expected amplicon of 263 bp. Genomic DNA and RNA with no reverse transcription step from both wild-type and mutant E. chaffeensis were included as controls, along with a no-template reaction (−). DNA marker (1 kb Plus DNA ladder) (L) was used to identify specific size amplicon. The asRNA amplicon was detected in the cDNA from the mutant, but not in the cDNA from wild type. Mutant genomic DNA served as the positive control and similarly genomic DNA from wild-type E. chaffeensis served as a negative control along with a no-template negative control. RNA-only controls (with no reverse transcription steps) also yielded the anticipated negative results. ( E ) The impact of ECH_1143 antisense construct insertion on the RNA expression of upstream ( ECH_0230 ) and downstream ( ECH_0232 ) genes. Transcriptional analysis of RNA isolated from asRNA mutant (M) and wild-type (W) E. chaffeensis in macrophage cells was conducted by RT-PCR targeting ECH_0230 and ECH_0232 . RNA input was normalized based on 16S rRNA levels quantified by qRT-PCR. Transcript levels of the target genes were then assessed by semi-quantitative RT-PCR (30 cycles). Wild-type genomic DNA served as a positive control (+), and a no-template reaction was included as a negative control (−). DNA marker (1 kb Plus DNA ladder) (L) was used to verify the expected amplicon sizes. Expression of ECH_0230 and ECH_0232 was similar for wild-type and mutant E. chaffeensis .

Article Snippet: Additionally, whole-genome sequencing was performed using mutant genomic DNA on Nanopore sequencing platform (Plasmidsaurus Inc., KY, USA) to independently confirm the mutation and to rule out any off-target insertions.

Techniques: Mutagenesis, Sequencing, Agarose Gel Electrophoresis, Amplification, Reverse Transcription Polymerase Chain Reaction, Isolation, Reverse Transcription, Marker, Positive Control, Negative Control, Construct, RNA Expression, Quantitative RT-PCR, Expressing

Journal: bioRxiv

Article Title: An improved CRISPR-base editor tool to target virulence factors in the ruminant pathogen Mycoplasma bovis

doi: 10.64898/2026.05.29.712936

Figure Lengend Snippet: (A) pFRIT4.0-DgRNA plasmid map. The two sgRNA cassettes are indicated (B) Genome positions of MBOVPG45_0215 ( mnuA ) and MBOVPG45_0690 (5’- nt ) targeted genes and respective location of the generated stop codons within genes. (C) Sanger sequencings of the targeted sites within MBOVPG45_0215 (left) and MBOVPG45_0690 (right) of an isolated mutant. The percentage of bases found in the isolated clones of transformants were determined from Sanger sequencing chromatograms using the Base editor processing tool from Han lab .

Article Snippet: Finally, whole plasmid sequencing was conducted through the use of the Plasmid-EZ service (Genewiz) or the Plasmidsaurus sequencing platform.

Techniques: Plasmid Preparation, Generated, Isolation, Mutagenesis, Clone Assay, Sequencing

Journal: bioRxiv

Article Title: An improved CRISPR-base editor tool to target virulence factors in the ruminant pathogen Mycoplasma bovis

doi: 10.64898/2026.05.29.712936

Figure Lengend Snippet: (A) Localisation of the 11 copies of IS Mbov1 within the genome of M. bovis PG45 strain. (B) Targeted regions of IS Mbov1 copies through double gRNA expression and the respective location of the generated stop codons (C) IS Mbov1 isoform sequence conservation of targeted regions (representation through muscle alignment). (D) gRNAs target sequences conservation within IS Mbov1 done through Web-logo . (E) Sanger sequencing of the targeted sites, Target 1 (left) and Target 2 (right) within an isolated mutant after three rounds of induction. Chromatograms were analysed using the Base editor processing tool from Han lab .

Article Snippet: Finally, whole plasmid sequencing was conducted through the use of the Plasmid-EZ service (Genewiz) or the Plasmidsaurus sequencing platform.

Techniques: Expressing, Generated, Sequencing, Isolation, Mutagenesis

Journal: bioRxiv

Article Title: An improved CRISPR-base editor tool to target virulence factors in the ruminant pathogen Mycoplasma bovis

doi: 10.64898/2026.05.29.712936

Figure Lengend Snippet: To obtain full deamination on all ISMbov1 coding sequences, three rounds of induction were necessary. An example of the profiles obtained after one, two or three rounds of deamination for one of the three clones is presented here. The first round was conducted as previous cultures were, with overnight induction, followed by gDNA extraction and Sanger screening. After primary analysis, which revealed that full deamination had not occurred, two supplementary passages under inducting conditions were performed and gDNA was extracted at each of them for PCR amplification and Sanger sequencing.

Article Snippet: Finally, whole plasmid sequencing was conducted through the use of the Plasmid-EZ service (Genewiz) or the Plasmidsaurus sequencing platform.

Techniques: Clone Assay, Extraction, Amplification, Sequencing

Journal: bioRxiv

Article Title: An improved CRISPR-base editor tool to target virulence factors in the ruminant pathogen Mycoplasma bovis

doi: 10.64898/2026.05.29.712936

Figure Lengend Snippet: (A) Targeted regions of MIP genes conducted with CRISPR-VQR variants. (B) Representation of Sanger sequencing of the targeted sites within MBOVPG45_0373 (left) or MBOVPG45_0376 (Right) within two isolated mutants. The black square indicates the used PAM sequence. Sanger sequencing chromatogram analysis was done using Base editor processing tool from Han lab .

Article Snippet: Finally, whole plasmid sequencing was conducted through the use of the Plasmid-EZ service (Genewiz) or the Plasmidsaurus sequencing platform.

Techniques: CRISPR, Sequencing, Isolation

Journal: bioRxiv

Article Title: An improved CRISPR-base editor tool to target virulence factors in the ruminant pathogen Mycoplasma bovis

doi: 10.64898/2026.05.29.712936

Figure Lengend Snippet: After recombination between the res sequences induced by the γδ resolvase, a 218 bp scar remains at the site of integration of the transposon. It includes the remaining res sequence (coloured in red) and inverted repeats (IR) at the extremities of the transposon (coloured in green).

Article Snippet: Finally, whole plasmid sequencing was conducted through the use of the Plasmid-EZ service (Genewiz) or the Plasmidsaurus sequencing platform.

Techniques: Sequencing

Journal: bioRxiv

Article Title: An improved CRISPR-base editor tool to target virulence factors in the ruminant pathogen Mycoplasma bovis

doi: 10.64898/2026.05.29.712936

Figure Lengend Snippet: The multi-step process for the generation of deaminated-resolved-cured (DRC) mutants involves first the transformation of the mycoplasma, the integration of the transposon harbouring the CRISPR-BE and induction of its expression to obtain a deaminated mutant. PCR and Sanger sequencing screening is then used to select a deaminated mutant. Resolution of the transposon is then achieved by transformation with the pRES plasmid. Continued passages of isolated mutants under selection with tetracycline 5 µg.µL - are combined with DNA extraction and PCR amplification at each passage to verify the excision of the transposon cassette. Once excision is validated by PCR, gentamicin susceptibility is confirmed by culture in gentamicin selective media at 100 µg.mL - . Following confirmation, the isolated mutants are cultivated in non-selective media and subcloned until negative PCR amplification of a pRES region and inability to grow in selective tetracycline medium are obtained. Final verifications were done through Sanger sequencing of the deamination site and WGS to verify genomic scar presence.

Article Snippet: Finally, whole plasmid sequencing was conducted through the use of the Plasmid-EZ service (Genewiz) or the Plasmidsaurus sequencing platform.

Techniques: Transformation Assay, CRISPR, Expressing, Mutagenesis, Sequencing, Plasmid Preparation, Isolation, Selection, DNA Extraction, Amplification

Journal: bioRxiv

Article Title: An improved CRISPR-base editor tool to target virulence factors in the ruminant pathogen Mycoplasma bovis

doi: 10.64898/2026.05.29.712936

Figure Lengend Snippet: WGS sequencing revealed the successful resolution of the transposon and indicated insertion locus of the employed CRISPR-BE was intragenic to the MBOVPG45_756 coding sequence. (A) Genome structure in the WT and in the resolved mutant. (B) Localisation of the transposon insertion site and targeted sites in the genome of M. bovis PG45.

Article Snippet: Finally, whole plasmid sequencing was conducted through the use of the Plasmid-EZ service (Genewiz) or the Plasmidsaurus sequencing platform.

Techniques: Sequencing, CRISPR, Mutagenesis

Journal: bioRxiv

Article Title: An improved CRISPR-base editor tool to target virulence factors in the ruminant pathogen Mycoplasma bovis

doi: 10.64898/2026.05.29.712936

Figure Lengend Snippet: In the case of the obtained MBOVPG45_0215 mutant with the pFrHog plasmid, the process to obtain a cured mutant is a condensed version of the previously described DRC pipeline. After transformation, a single round of induction is necessary to obtain the desired base-editing. PCR verifications for the presence of mutation and cultures in selective medium are used to follow the process. Curing of the plasmid is then obtained by passages in non-selective medium. Once both validation steps are validated, the isolated mutant is considered to be cured and can be reverified at the deamination site through PCR and Sanger sequencing.

Article Snippet: Finally, whole plasmid sequencing was conducted through the use of the Plasmid-EZ service (Genewiz) or the Plasmidsaurus sequencing platform.

Techniques: Mutagenesis, Plasmid Preparation, Transformation Assay, Biomarker Discovery, Isolation, Sequencing

Journal: bioRxiv

Article Title: An improved CRISPR-base editor tool to target virulence factors in the ruminant pathogen Mycoplasma bovis

doi: 10.64898/2026.05.29.712936

Figure Lengend Snippet: Representation of the percentage of targetable CDS by the CRISPR-BE or CRISPR-BE VQR tools compared to the total number of CDS in multiple mycoplasma species. Representation of the percentage of genes targetable within the first 10 th , 30 th , 50 th and whole coding sequence.

Article Snippet: Finally, whole plasmid sequencing was conducted through the use of the Plasmid-EZ service (Genewiz) or the Plasmidsaurus sequencing platform.

Techniques: CRISPR, Sequencing

Journal: Frontiers in Oncology

Article Title: Beyond counting: how single-cell long-read sequencing turns transcriptome complexity into precision targets

doi: 10.3389/fonc.2026.1800370

Figure Lengend Snippet: Single-cell long-read sequencing (scLRS) enhances tumor cell resolution by integrating methods for detecting splicing alterations and genetic variants. Schematic representation of how scLRS can visualize and combine multiple information types within the same cells to improve tumor cell resolution. Created in BioRender. Byrne, A. (2026) https://BioRender.com/mrmobm6 .

Article Snippet: Adopting third-generation long-read sequencing platforms, such as those developed by Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT), offers a solution to overcome this limitation and deepen our understanding of the transcriptome.

Techniques: Single Cell, Sequencing