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: Nature Communications

Article Title: FOXD1-dependent MICU1 expression regulates mitochondrial activity and cell differentiation

doi: 10.1038/s41467-018-05856-4

Figure Lengend Snippet: MICU1 is differentially expressed during embryonic development. a – c Representative western blot for lysates from brain ( a ), heart ( b ), and liver ( c ) harvested from embryo (E13.5), and neonates (P0, P2, and P4). The western blots were probed with antibody specific for MCU, MICU1, MCUR1, and Tom20. d – f Quantification of relative MCU, MICU1, and MCUR1 protein abundance from a – c . g Representative zoomed microscopic images of the tissue microarray probed with MICU1 antibody. h Quantification of MICU1 expression levels represented as percent MICU1 expression as a function of tissue staining. i Representative western blot for lysates from control fibroblasts (GM942), hiPSCs (SeV3, SeV5, and SV20), and hESCs probed with antibody specific for MCU, MICU1, Tom20, and β-actin. j Quantification of relative MICU1 protein abundance from i . k Quantification of MICU1 mRNA levels in different tissues (brain, liver, and heart) harvested from embryo and neonates (E13.5, P0, P2, and P4). l Quantification of MICU1 mRNA levels in two independent clonal hiPSCs lines (SeV3 and SV20) and one clonal line of SeV3 (SeV5). Data represents Mean ± SEM; ** P < 0.01; *** P < 0.001; n = 3–5 (one-way ANOVA)

Article Snippet: Light Switch Promoter Reporter GoClone plasmid with MICU1 promoter sequence cloned (SwitchGear Genomics; S712264) and the corresponding control plasmids were used for luciferase assay.

Techniques: Western Blot, Microarray, Expressing, Staining

Journal: Nature Communications

Article Title: FOXD1-dependent MICU1 expression regulates mitochondrial activity and cell differentiation

doi: 10.1038/s41467-018-05856-4

Figure Lengend Snippet: MICU1 is transcriptionally regulated by Foxd1 under hypoxia. a Quantification of MICU1 mRNA abundance in hiPSCs exposed to normoxia (20% O 2 ) and hypoxia (5% O 2 ). b Representative western blot for lysates from control fibroblasts and hiPSCs grown under hypoxic/normoxic conditions and probed with antibody specific for MICU1, MCU, ATP5A, and Tom20. c , d Quantification of relative MICU1 ( c ) and MCU ( d ) protein abundance in hiPSCs grown under hypoxic/normoxic conditions. e Quantification of Foxd1 mRNA abundance in hiPSCs under normoxia/hypoxia. f Representative western blot for lysates from control fibroblasts and hiPSCs grown under normoxic/ hypoxic conditions and probed with antibody specific for Foxd1 and β-actin. g Quantification of relative protein abundance of Foxd1 and MICU1 quantified from f and b . h Quantification of Foxd1 mRNA abundance in various tissues (brain, heart, liver, and lung) harvested from different stages of embryonic development. i Representative western blot for lysates from brain, heart, liver, and lung harvested from embryos/neonates and probed with antibodies specific for MICU1, Foxd1, and β-actin. j Quantification of relative protein abundance of Foxd1 and MICU1 quantified from i . k ChIP-assay was performed in hiPSCs exposed to normoxia and hypoxia. Antibody specific for Foxd1 was used to immunoprecipitate the chromatin and the fold enrichment of micu1 promoter relative to the matched input control was quantified by qRT-PCR. Bar represents Mean ± SEM, ** P < 0.01, * P < 0.05, *** P < 0.001; n = 3–5 (One-Way ANOVA)

Article Snippet: Light Switch Promoter Reporter GoClone plasmid with MICU1 promoter sequence cloned (SwitchGear Genomics; S712264) and the corresponding control plasmids were used for luciferase assay.

Techniques: Western Blot, Quantitative RT-PCR

Journal: Nature Communications

Article Title: FOXD1-dependent MICU1 expression regulates mitochondrial activity and cell differentiation

doi: 10.1038/s41467-018-05856-4

Figure Lengend Snippet: Loss of MICU1 in hiPSCs augment MCU-mediated Ca 2+ uptake and overloads matrix Ca 2+ . An equal number of fibroblasts (GM942), hiPSCs (SeV3), and SeV3 expressing MICU1cells (6 × 10 6 ) were permeabilized with digitonin (40 μM), loaded with JC-1 (800 nM) and Fura-FF (1 μM) and changes in Δ Ψ m and m Ca 2+ uptake was measured using a multi-wavelength excitation dual-wavelength emission spectrofluorimeter. a Mean traces of Δ Ψ m in fibroblasts and hiPSCs. b Quantification of basal Δ Ψ m before addition of an extramitochondrial Ca 2+ bolus. c Mean traces of [Ca 2+ ] out measured in permeabilized fibroblasts and hiPSCs. d Quantification of the rate of m Ca 2+ uptake as a function of decrease in bath Ca 2+ fluorescence after an extramitochondrial Ca 2+ pulse (1 μM). e Mean traces of [Ca 2+ ] out after addition of CCCP. f Quantification of total m Ca 2+ released after the addition of CCCP as a function of increase in bath Ca 2+ fluorescence. Foxd1 knock down restores MICU1 gate keeping effect. g – k SeV3 cells were transiently transfected with control and Foxd1 siRNA and 48 h post transfection, Δ Ψ m and m Ca 2+ uptake was measured simultaneously in permeabilized cells. g Mean traces of Δ Ψ m in control (Neg siRNA) and Foxd1 KD hiPSCs. h Mean traces of [Ca 2+ ] out measured in permeabilized control (Neg siRNA) and Foxd1 KD hiPSCs. i Quantification of basal Δ Ψ m before addition of an extramitochondrial Ca 2+ bolus. j Quantification of the rate of m Ca 2+ uptake as a function of decrease in bath Ca 2+ fluorescence after an extramitochondrial Ca 2+ pulse (1 μM). k Quantification of total m Ca 2+ released after the addition of CCCP. Data represents Mean ± SEM; ** P < 0.01, *** P < 0.001; n = 4–6 (One-way ANOVA)

Article Snippet: Light Switch Promoter Reporter GoClone plasmid with MICU1 promoter sequence cloned (SwitchGear Genomics; S712264) and the corresponding control plasmids were used for luciferase assay.

Techniques: Expressing, Fluorescence, Transfection

Journal: Nature Communications

Article Title: FOXD1-dependent MICU1 expression regulates mitochondrial activity and cell differentiation

doi: 10.1038/s41467-018-05856-4

Figure Lengend Snippet: Reconstitution of MICU1 in hiPSCs establishes i Ca 2+ transients and modulates lineage-specific cell differentiation. a Representative western blot for lysates from hiPSCs with and without MICU1 expression. The western blots were probed with antibody specific for MICU1, MCU, ATP5A, Cytochrome C, Foxd1, Tom20, and β-actin. b Quantification of relative protein abundance of Foxd1 and MICU1. c Traces of spontaneous cytosolic Ca 2+ oscillations in control fibroblasts and hiPSCs with and without MICU1 expression. Intact cells were loaded with fluo4 and spontaneous Ca 2+ oscillations were observed at 488 nm. d Quantification of peak fluo-4 fluorescence. e Quantification of the frequency of c Ca 2+ oscillations. f Representative western blot for lysates from hiPSCs induced for lineage specific differentiation for the specified days with and without MICU1 expression. The western blots were probed with antibody specific for MICU1, MCU, Foxd1, and β-actin. g Traces of spontaneous c Ca 2+ oscillations in hiPSCs upon induction of lineage specific differentiation with and without MICU1 expression. Ca 2+ transients were measured at different days of differentiation: Day 3, 4, and 5 for endoderm and mesoderm; Day 5, 6, and 7 for ectoderm. h Quantification of peak fluo-4 fluorescence. i Quantification of the frequency of c Ca 2+ oscillations. Data indicate quantified peak fluorescence/frequency of oscillation. Mean ± SEM is represented in the figures; ** P < 0.01, * P < 0.05, *** P < 0.001; n = 3–4 (One-Way Anova) individual experiments. Dots represents mean data from group of single cells

Article Snippet: Light Switch Promoter Reporter GoClone plasmid with MICU1 promoter sequence cloned (SwitchGear Genomics; S712264) and the corresponding control plasmids were used for luciferase assay.

Techniques: Cell Differentiation, Western Blot, Expressing, Fluorescence

Journal: Nature Communications

Article Title: FOXD1-dependent MICU1 expression regulates mitochondrial activity and cell differentiation

doi: 10.1038/s41467-018-05856-4

Figure Lengend Snippet: MICU1 expression promotes iPSC-Cardiomyocyte maturation. a Representative western blot for lysates from SeV3 and SeV3-derived cardiomyocytes (SeV3-CMs) with and without MICU1 expression. The western blots were probed with antibody specific for MICU1, MCU, Foxd1, Tom20, and β-actin. b Quantification of the MICU1 mRNA abundance in SeV3 and day 24 SeV3-CMs. c Traces of spontaneous cytosolic Ca 2+ oscillations in SeV3-CMs with and without MICU1 expression at day 24 and day 46 post-differentiation. d Quantification of peak fluo-4 fluorescence. e Quantification of the frequency of c Ca 2+ oscillations. Data indicate Mean ± SEM; ** P < 0.001, *** P < 0.001; n = 3–4 independent experiments. Dots represents data from group of single cells. f Quantification of mRNA abundance of the early cardiac transcription factor Mef2c in SeV3-CMs with or without MICU1 expression at day 24 of differentiation. g Quantification of mRNA abundance of the cardiac contractility proteins, MYH6, and MYH7 in SeV3-CMs with or without MICU1 expression at day 24 of differentiation. Data indicate Mean ± SEM; * P < 0.05, *** P < 0.001; n = 3–4. h , j Measurement of oxygen consumption rate (OCR) in SeV3-CMs with or without MICU1 expression using glucose (glucose oxidation) ( h ) or carnitine (fatty acid oxidation) ( j ) as substrate (glucose oxidation). After basal OCR measurement, oligomycin (A), FCCP (B), and rotenone + Antimycin A (C) were added as indicated. h , j Representative traces of OCR in SeV3-CMs. i , k Quantification of basal and maximal OCR, and spare capacity in SeV3-CMs. Data indicate Mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.0001; n = 3

Article Snippet: Light Switch Promoter Reporter GoClone plasmid with MICU1 promoter sequence cloned (SwitchGear Genomics; S712264) and the corresponding control plasmids were used for luciferase assay.

Techniques: Expressing, Western Blot, Derivative Assay, Fluorescence