Journal: Frontiers in Microbiology

Article Title: Rescue of Recombinant Adenoviruses by CRISPR/Cas-Mediated in vivo Terminal Resolution

doi: 10.3389/fmicb.2022.854690

Figure Lengend Snippet: (A) Shown is the fold increase in the mean fluorescent intensity (MFI) in Flag-tag-specific FACS measurements of different 293A-Cas9 clones expressing Flag-tagged Cas9 compared to the parental 293A signal. Data shown represent the mean of two independent experiments, with error bars indicating the range of measurements. All presented clones were also checked for CTR competency of rAd rescue by co-transfecting of pBWH-C5-mChe and pAR-gRNA-Ex. Clones that appeared to be permissive for rAd rescue based on two experiments are indicated by orange bars, while the white bars indicate clones, which appeared to be non-permissive in at least one experiment. (B) Comparison of different approaches for supplying necessary components for CRISPR/Cas9-mediated CTR. Co-transfection of helper plasmid(s) (Helper) expressing sgRNA and Cas9 protein with the construct carrying the ACT flanked rAd bacmids (rAd, red bar, this approach was applied for most of the experiments described in the manuscript) was compared to combination of all CTR components in one construct coding for the rAd genome and all necessary CRISPR/Cas9-components in the vector backbone (purple bar); the Cas9 is delivered by constitutive expression in the cell line B2 or b5 [see (A) ] used to rescue the rAd, while a bacmid coding for a rAd genome is co-transfected together with a sgRNA-expressing plasmid as in panel (A) (dark and light orange bars); and the same as in previously, but sgRNA is expressed from the same construct that carries the rAd (dark and light gray bars). The primary rescue efficiencies were obtained as in . Significance was calculated using Welch’s ANOVA test.

Article Snippet: The expression cassette coding for the destabilized, human codon-optimized Cas9 fusion protein (DD-Cas9) was constructed on the basis of pDD-Cas9 [Addgene Plasmid #90086, a kind gift from Raffaella Sordella ( )] by inserting a glutamine codon instead of the first methionine codon of the Cas9 coding sequence.

Techniques: FLAG-tag, Clone Assay, Expressing, CRISPR, Cotransfection, Plasmid Preparation, Construct, Transfection

Journal: Frontiers in Microbiology

Article Title: Rescue of Recombinant Adenoviruses by CRISPR/Cas-Mediated in vivo Terminal Resolution

doi: 10.3389/fmicb.2022.854690

Figure Lengend Snippet: ITR-near CRISPR-Cas-mediated cleavage increased the efficient of recombinant adenoviruses rescue. (A) Another sgRNA (sgRNA-Int5, purple, PAM underlined) targeting the ITRs (bold) can induce Cas9-mediated cleavage at the ITRs (purple triangles). To check the impact of cleavage distance, the ITRs were extended by CRISPR/Cas9 target sequences ( ACT , red) using a 12-bp long spacer (black underlined), which was targeted by sgRNA-Ex (red, PAM is underlined) inducing double-strand breaks (red triangles) 18–19 bp upstream of the ITRs. (B) rAd reconstitution efficiencies were compared after co-transfection of 293A cells with pBWH-C5-mChe and pSG5-Cas9F in the presence of either sgRNA-Int5 (Ad5-Int5) or sgRNA-Ex (Ad5-Ex), and with pBWH18/19-C5-mChe in the presence of sgRNA-Ex (Ad518/19-Ex). The primary rescue efficiencies were obtained as in . Significance was calculated using Welch ANOVA test. **** p < 0.0001.

Article Snippet: The expression cassette coding for the destabilized, human codon-optimized Cas9 fusion protein (DD-Cas9) was constructed on the basis of pDD-Cas9 [Addgene Plasmid #90086, a kind gift from Raffaella Sordella ( )] by inserting a glutamine codon instead of the first methionine codon of the Cas9 coding sequence.

Techniques: CRISPR, Recombinant, Cotransfection

Journal: Frontiers in Microbiology

Article Title: Rescue of Recombinant Adenoviruses by CRISPR/Cas-Mediated in vivo Terminal Resolution

doi: 10.3389/fmicb.2022.854690

Figure Lengend Snippet: ITR-near CTR induced also efficient rescue of recombinant adenoviruses based on HAdV-4. (A) Similarly to the HAdV-5-based constructs (upper panel), a recombinant HAdV-4 based bacmid was constructed (lower panel), which was flanked with ACT sequences (red, PAM is underlined) at both ITRs (here, the right ITR is shown, white letters). This sequence can be targeted by the universal sgRNA-Ex, like for the HAdV-5 based constructs (upper panel). Here also another sgRNA (sgRNA-Int4, blue, PAM is underlined) targeting the HAdV-4 ITRs (bold) can induce Cas9-mediated cleavage at the ITRs (blue triangles). (B) The rAd reconstitution efficiencies were determined after co-transfection of A549, SKOV-3, and 293A cells with pBWH-E4-ΔE3 with either pAR-gRNA-Cas9F-Amp expressing sgRNA-Ex (E4-Ex) or with pAR-Int4-Cas9F-Amp (E4-Int4, for 293A cells only). The primary rescue efficiencies were obtained as in . A549-Ex and SKOV-3-Ex were tested twice in technical duplicates, and error bars represent spread of results. E4-Ex and E4-Int4 were done three times in technical duplicates, and error bars represent standard deviation. Significance was calculated using unpaired t -test comparing E4-Ex and E4-Int4. ** p < 0.01.

Article Snippet: The expression cassette coding for the destabilized, human codon-optimized Cas9 fusion protein (DD-Cas9) was constructed on the basis of pDD-Cas9 [Addgene Plasmid #90086, a kind gift from Raffaella Sordella ( )] by inserting a glutamine codon instead of the first methionine codon of the Cas9 coding sequence.

Techniques: Recombinant, Construct, Sequencing, Cotransfection, Expressing, Standard Deviation

Journal: Frontiers in Microbiology

Article Title: Rescue of Recombinant Adenoviruses by CRISPR/Cas-Mediated in vivo Terminal Resolution

doi: 10.3389/fmicb.2022.854690

Figure Lengend Snippet: CRISPR-Cas-mediated cleavage in cells yielded efficient rescue of recombinant adenoviruses based on HAdV-5. (A) The ITRs on an existing rAd-plasmid were extended by artificial CRISPR/Cas9 target sequences ( ACT sequences , red), which were targeted by sgRNA-Ex (red line, PAM is underlined) inducing double-strand breaks 6–7 bp outside of the ITRs (indicated by the red triangles on the sgRNA targeting strand only). (B) Reconstitution efficiency of rAds in 293A cells after transfection of circular pBWH-C5-mChe (an ACT-flanked Ad5-bacmid) alone (cir5), a linearized rAd5 bacmid (lin5), a circular rAd5 bacmid carrying fusion ITRs (ITRf 5) or co-transfection of circular pBWH-C5-mChe with pAR-gRNA-Cas9F-Amp, a Cas9, and gRNA-Ex expressing helper plasmid for CRISPR/Cas-mediated terminal resolution (CTR 5). The appearing plaques were counted after seeding the transfectants into multiwell plates by observing the plates for 14 days after transfection. Statistical significance was determined using Welch ANOVA tests. (C) The rAd reconstitution efficiencies (as foci/μg DNA) in 293A cells of pBWH-C5-mChe-DD-Cas9, expressing conditional Cas9 (green bars) with co-expression of sgRNA-Ex, in the presence of stabilizer Shield-1 at the indicated concentrations are compared to the efficiency of CTR using Cas9F as for CTR 5 in (B) (red bar). The primary rescue efficiencies were obtained as in (B) . Significance was calculated using ordinary one-way ANOVA. * p < 0.05 and ** p < 0.01.

Article Snippet: The expression cassette coding for the destabilized, human codon-optimized Cas9 fusion protein (DD-Cas9) was constructed on the basis of pDD-Cas9 [Addgene Plasmid #90086, a kind gift from Raffaella Sordella ( )] by inserting a glutamine codon instead of the first methionine codon of the Cas9 coding sequence.

Techniques: CRISPR, Recombinant, Plasmid Preparation, Transfection, Cotransfection, Expressing

Journal: Molecular microbiology

Article Title: The Regulation of Antimicrobial Peptide Resistance in the Transition to Insect Symbiosis

doi: 10.1111/mmi.13598

Figure Lengend Snippet: Distribution and sequence coverage of Tn5 mutants in the S. praecaptivus genome are depicted as a histogram (A, Upper). Note that mutations affecting Sant_4061 are the most abundant in the library, following growth in LB media. The zoomed in regions of the histogram (A, Lower) reveal the distribution and abundance of Tn5 mutants in the genomic regions encoding phoPQ and Sant_4061 (in red), with the mutants recovered in the screen for polymyxin B sensitivity highlighted in orange, with the number of recovered mutants shown above each column. Growth curves for WT S. praecaptivus and Sant_4061 and phoP mutants grown in either LB or LB + Polymyxin B (PB) for 10 hours (B). Standard errors are shown above and below the average of three replicates at each time point. ΔSant_4061, ΔphoQ, and ΔphoP are unable to grow on LB supplemented with polymyxin B (C).

Article Snippet: Construction of phoP and phoQ knockouts A plasmid construct consisting of approximately 500 bp of homologous sequence up and downstream of phoP with an internal kanamycin cassette was commercially produced (Genscript).

Techniques: Sequencing

Journal: Molecular microbiology

Article Title: The Regulation of Antimicrobial Peptide Resistance in the Transition to Insect Symbiosis

doi: 10.1111/mmi.13598

Figure Lengend Snippet: Phylogeny of S. praecaptivus and related Sodalis-allied endosymbionts and free-living bacteria based on maximum likelihood analyses of the phoQ coding sequence (1.45 kbp) and 16S rRNA (1.46 kbp). Sequences below strain names on the phoQ phylogeny show the amino acids sequences of the Mg2+-binding site within PhoQ, with acidic residues highlighted in bold. The numbers adjacent to nodes indicate maximum likelihood bootstrap values shown for nodes with bootstrap support > 80%.

Article Snippet: Construction of phoP and phoQ knockouts A plasmid construct consisting of approximately 500 bp of homologous sequence up and downstream of phoP with an internal kanamycin cassette was commercially produced (Genscript).

Techniques: Bacteria, Sequencing, Binding Assay

Journal: Molecular microbiology

Article Title: The Regulation of Antimicrobial Peptide Resistance in the Transition to Insect Symbiosis

doi: 10.1111/mmi.13598

Figure Lengend Snippet: Bacterial strains used in this study

Article Snippet: Construction of phoP and phoQ knockouts A plasmid construct consisting of approximately 500 bp of homologous sequence up and downstream of phoP with an internal kanamycin cassette was commercially produced (Genscript).

Techniques: