plasmid sequence Search Results


90085 deposited by raffaella sordella lab 52  (Addgene inc)
93
Addgene inc 90085 deposited by raffaella sordella lab 52
90085 Deposited By Raffaella Sordella Lab 52, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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90085 deposited by raffaella sordella lab 52 - by Bioz Stars, 2026-05
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fragment 1  (Addgene inc)
93
Addgene inc fragment 1
Fragment 1, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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fragment 1 - by Bioz Stars, 2026-05
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cas9 fusion protein dd cas9  (Addgene inc)
93
Addgene inc cas9 fusion protein dd cas9
(A) Shown is the fold increase in the mean fluorescent intensity (MFI) in Flag-tag-specific FACS measurements of different <t>293A-Cas9</t> 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 <t>Cas9</t> <t>protein</t> 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.
Cas9 Fusion Protein Dd Cas9, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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cas9 fusion protein dd cas9 - by Bioz Stars, 2026-05
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plasmid sequences  (Addgene inc)
94
Addgene inc plasmid sequences
(A) Shown is the fold increase in the mean fluorescent intensity (MFI) in Flag-tag-specific FACS measurements of different <t>293A-Cas9</t> 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 <t>Cas9</t> <t>protein</t> 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.
Plasmid Sequences, supplied by Addgene inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/plasmid sequences/product/Addgene inc
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plasmid sequences - by Bioz Stars, 2026-05
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plasmid dna sequencing  (Eurofins)
90
Eurofins plasmid dna sequencing
(A) Shown is the fold increase in the mean fluorescent intensity (MFI) in Flag-tag-specific FACS measurements of different <t>293A-Cas9</t> 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 <t>Cas9</t> <t>protein</t> 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.
Plasmid Dna Sequencing, supplied by Eurofins, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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plasmid dna sequencing - by Bioz Stars, 2026-05
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plasmid dna sequencing  (ACGT Inc)
90
ACGT Inc plasmid dna sequencing
(A) Shown is the fold increase in the mean fluorescent intensity (MFI) in Flag-tag-specific FACS measurements of different <t>293A-Cas9</t> 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 <t>Cas9</t> <t>protein</t> 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.
Plasmid Dna Sequencing, supplied by ACGT Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/plasmid dna sequencing/product/ACGT Inc
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plasmid dna sequencing - by Bioz Stars, 2026-05
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plasmid containing an endopeptidase-negative (i.e. non-toxic) bont/e dna sequence  (Ipsen Group)
90
Ipsen Group plasmid containing an endopeptidase-negative (i.e. non-toxic) bont/e dna sequence
(A) Shown is the fold increase in the mean fluorescent intensity (MFI) in Flag-tag-specific FACS measurements of different <t>293A-Cas9</t> 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 <t>Cas9</t> <t>protein</t> 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.
Plasmid Containing An Endopeptidase Negative (I.E. Non Toxic) Bont/E Dna Sequence, supplied by Ipsen Group, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/plasmid containing an endopeptidase-negative (i.e. non-toxic) bont/e dna sequence/product/Ipsen Group
Average 90 stars, based on 1 article reviews
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lentiviral plasmids containing hspb6 small hairpin rna (shrna) sequences  (Genechem)
90
Genechem lentiviral plasmids containing hspb6 small hairpin rna (shrna) sequences
(A) Shown is the fold increase in the mean fluorescent intensity (MFI) in Flag-tag-specific FACS measurements of different <t>293A-Cas9</t> 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 <t>Cas9</t> <t>protein</t> 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.
Lentiviral Plasmids Containing Hspb6 Small Hairpin Rna (Shrna) Sequences, supplied by Genechem, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 90 stars, based on 1 article reviews
lentiviral plasmids containing hspb6 small hairpin rna (shrna) sequences - by Bioz Stars, 2026-05
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plasmid containing ctsk coding sequence (cds)  (Shanghai GenePharma)
90
Shanghai GenePharma plasmid containing ctsk coding sequence (cds)
(A) Shown is the fold increase in the mean fluorescent intensity (MFI) in Flag-tag-specific FACS measurements of different <t>293A-Cas9</t> 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 <t>Cas9</t> <t>protein</t> 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.
Plasmid Containing Ctsk Coding Sequence (Cds), supplied by Shanghai GenePharma, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/plasmid containing ctsk coding sequence (cds)/product/Shanghai GenePharma
Average 90 stars, based on 1 article reviews
plasmid containing ctsk coding sequence (cds) - by Bioz Stars, 2026-05
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plasmid sequencing  (Sangon Biotech)
90
Sangon Biotech plasmid sequencing
(A) Shown is the fold increase in the mean fluorescent intensity (MFI) in Flag-tag-specific FACS measurements of different <t>293A-Cas9</t> 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 <t>Cas9</t> <t>protein</t> 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.
Plasmid Sequencing, supplied by Sangon Biotech, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/plasmid sequencing/product/Sangon Biotech
Average 90 stars, based on 1 article reviews
plasmid sequencing - by Bioz Stars, 2026-05
90/100 stars
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plasmid purification and sequencing  (GATC Biotech)
90
GATC Biotech plasmid purification and sequencing
(A) Shown is the fold increase in the mean fluorescent intensity (MFI) in Flag-tag-specific FACS measurements of different <t>293A-Cas9</t> 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 <t>Cas9</t> <t>protein</t> 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.
Plasmid Purification And Sequencing, supplied by GATC Biotech, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/plasmid purification and sequencing/product/GATC Biotech
Average 90 stars, based on 1 article reviews
plasmid purification and sequencing - by Bioz Stars, 2026-05
90/100 stars
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plasmid dna sequences  (Operon Biotech)
90
Operon Biotech plasmid dna sequences
(A) Shown is the fold increase in the mean fluorescent intensity (MFI) in Flag-tag-specific FACS measurements of different <t>293A-Cas9</t> 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 <t>Cas9</t> <t>protein</t> 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.
Plasmid Dna Sequences, supplied by Operon Biotech, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/plasmid dna sequences/product/Operon Biotech
Average 90 stars, based on 1 article reviews
plasmid dna sequences - by Bioz Stars, 2026-05
90/100 stars
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Image Search Results


(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.

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

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.

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

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.

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

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.

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