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wild type acriia4  (Addgene inc)


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    Addgene inc wild type acriia4
    Fig. 1. Kinetic insulation of CRISPR ON- and OFF-target effects by coexpression of anti-CRISPR proteins. (A) Schematic of a model for Cas9 genome editing. After cotransfec- tion with plasmids encoding Cas9 and sgRNA, plasmids are transcribed to Cas9-mRNA and sgRNA or degraded. Furthermore, the model describes the turnover of mRNAs, sgRNA, Cas9 protein, binding of sgRNA and Cas9, association of Cas9:sgRNA with the target gene, and gene editing. DNAsite, unedited target locus; DNAedited, edited target locus; D:sgR:C, trimeric complex of DNA, sgRNA, and Cas9. (B) Modeling of editing kinetics at high-affinity (ON-target) and low-affinity (OFF-target) sites. Left: The model describes concentrations of the gRNA and Cas9 over time after transient transfection and relates sgRNA and Cas9 expression to a gene-modified fraction of cells. The final gene-edited fraction depends on the integral of Cas9:sgRNA complex expression (upper left panel). Right: Relation between editing efficiency and Cas9 activity (time integral of Cas9:sgRNA complex). The target affinity of an sgRNA determines the editing efficiency at a respective locus. At very large Cas9:sgRNA integrals, gene-edited fractions reach saturation, irrespective of the target affinity. (C) Schematic of constructs used for expression of Cas9, <t>AcrIIA4,</t> and sgRNAs. NLS, nuclear localization signal. (D and E) Coexpressing mild doses of AcrIIA4 improves genome editing specificity. Cells were cotransfected with plasmids encoding AcrIIA4, Cas9, and an sgRNA targeting the AAVS1 locus and incubated for 72 hours followed by T7 endonuclease assay. The AcrIIA4 vector dose used during transfection is indicated. Twenty-two nanograms thereby corresponds to a threefold excess of Cas9/ sgRNA vectors. (D) Representative gel image and (E) quantification of InDel frequencies. (F) HEK 293T cells were cotransduced with 33 l of Cas9 AAV, 33 l of sgRNA AAV, and the indicated volume of AcrIIA4 AAV on two consecutive days. Volumes correspond to the amount of AAV-containing cell lysate applied (see Materials and Methods). Cells were incu- bated for 72 hours followed by T7 endonuclease assay. (E and F) Bars indicate mean editing frequencies; dots are individual data points from n = 3 independent experiments.
    Wild Type Acriia4, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    wild type acriia4 - by Bioz Stars, 2026-09
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    1) Product Images from "Coupling Cas9 to artificial inhibitory domains enhances CRISPR-Cas9 target specificity."

    Article Title: Coupling Cas9 to artificial inhibitory domains enhances CRISPR-Cas9 target specificity.

    Journal: Science advances

    doi: 10.1126/sciadv.aay0187

    Fig. 1. Kinetic insulation of CRISPR ON- and OFF-target effects by coexpression of anti-CRISPR proteins. (A) Schematic of a model for Cas9 genome editing. After cotransfec- tion with plasmids encoding Cas9 and sgRNA, plasmids are transcribed to Cas9-mRNA and sgRNA or degraded. Furthermore, the model describes the turnover of mRNAs, sgRNA, Cas9 protein, binding of sgRNA and Cas9, association of Cas9:sgRNA with the target gene, and gene editing. DNAsite, unedited target locus; DNAedited, edited target locus; D:sgR:C, trimeric complex of DNA, sgRNA, and Cas9. (B) Modeling of editing kinetics at high-affinity (ON-target) and low-affinity (OFF-target) sites. Left: The model describes concentrations of the gRNA and Cas9 over time after transient transfection and relates sgRNA and Cas9 expression to a gene-modified fraction of cells. The final gene-edited fraction depends on the integral of Cas9:sgRNA complex expression (upper left panel). Right: Relation between editing efficiency and Cas9 activity (time integral of Cas9:sgRNA complex). The target affinity of an sgRNA determines the editing efficiency at a respective locus. At very large Cas9:sgRNA integrals, gene-edited fractions reach saturation, irrespective of the target affinity. (C) Schematic of constructs used for expression of Cas9, AcrIIA4, and sgRNAs. NLS, nuclear localization signal. (D and E) Coexpressing mild doses of AcrIIA4 improves genome editing specificity. Cells were cotransfected with plasmids encoding AcrIIA4, Cas9, and an sgRNA targeting the AAVS1 locus and incubated for 72 hours followed by T7 endonuclease assay. The AcrIIA4 vector dose used during transfection is indicated. Twenty-two nanograms thereby corresponds to a threefold excess of Cas9/ sgRNA vectors. (D) Representative gel image and (E) quantification of InDel frequencies. (F) HEK 293T cells were cotransduced with 33 l of Cas9 AAV, 33 l of sgRNA AAV, and the indicated volume of AcrIIA4 AAV on two consecutive days. Volumes correspond to the amount of AAV-containing cell lysate applied (see Materials and Methods). Cells were incu- bated for 72 hours followed by T7 endonuclease assay. (E and F) Bars indicate mean editing frequencies; dots are individual data points from n = 3 independent experiments.
    Figure Legend Snippet: Fig. 1. Kinetic insulation of CRISPR ON- and OFF-target effects by coexpression of anti-CRISPR proteins. (A) Schematic of a model for Cas9 genome editing. After cotransfec- tion with plasmids encoding Cas9 and sgRNA, plasmids are transcribed to Cas9-mRNA and sgRNA or degraded. Furthermore, the model describes the turnover of mRNAs, sgRNA, Cas9 protein, binding of sgRNA and Cas9, association of Cas9:sgRNA with the target gene, and gene editing. DNAsite, unedited target locus; DNAedited, edited target locus; D:sgR:C, trimeric complex of DNA, sgRNA, and Cas9. (B) Modeling of editing kinetics at high-affinity (ON-target) and low-affinity (OFF-target) sites. Left: The model describes concentrations of the gRNA and Cas9 over time after transient transfection and relates sgRNA and Cas9 expression to a gene-modified fraction of cells. The final gene-edited fraction depends on the integral of Cas9:sgRNA complex expression (upper left panel). Right: Relation between editing efficiency and Cas9 activity (time integral of Cas9:sgRNA complex). The target affinity of an sgRNA determines the editing efficiency at a respective locus. At very large Cas9:sgRNA integrals, gene-edited fractions reach saturation, irrespective of the target affinity. (C) Schematic of constructs used for expression of Cas9, AcrIIA4, and sgRNAs. NLS, nuclear localization signal. (D and E) Coexpressing mild doses of AcrIIA4 improves genome editing specificity. Cells were cotransfected with plasmids encoding AcrIIA4, Cas9, and an sgRNA targeting the AAVS1 locus and incubated for 72 hours followed by T7 endonuclease assay. The AcrIIA4 vector dose used during transfection is indicated. Twenty-two nanograms thereby corresponds to a threefold excess of Cas9/ sgRNA vectors. (D) Representative gel image and (E) quantification of InDel frequencies. (F) HEK 293T cells were cotransduced with 33 l of Cas9 AAV, 33 l of sgRNA AAV, and the indicated volume of AcrIIA4 AAV on two consecutive days. Volumes correspond to the amount of AAV-containing cell lysate applied (see Materials and Methods). Cells were incu- bated for 72 hours followed by T7 endonuclease assay. (E and F) Bars indicate mean editing frequencies; dots are individual data points from n = 3 independent experiments.

    Techniques Used: Insulation, CRISPR, Protein Binding, Transfection, Expressing, Modification, Activity Assay, Construct, Incubation, Plasmid Preparation

    Fig. 2. Cas-Acr fusion design improves genome editing specificity. (A) Schematic of Cas-Acr constructs comprising Cas9 fused to an artificially weakened AcrIIA4 variant functioning as autoinhibitory domain (AID). (B to G) Cells were cotransfected with plasmids encoding the indicated Cas-Acr variant and an sgRNA targeting the AAVS1 (B and C), EMX1 (D and E), and HEK (F and G) locus and incubated for 72 hours followed by T7 endonuclease assay. Representative gel images (B, D, and F) and corresponding quantification of InDel frequencies (C, E, and G). Data are means ± SD; dots are individual data points from n = 3 independent experiments. Ins. 5, insertion variant 5 (see table S2); wt, Cas9 fused to wild-type AcrIIA4.
    Figure Legend Snippet: Fig. 2. Cas-Acr fusion design improves genome editing specificity. (A) Schematic of Cas-Acr constructs comprising Cas9 fused to an artificially weakened AcrIIA4 variant functioning as autoinhibitory domain (AID). (B to G) Cells were cotransfected with plasmids encoding the indicated Cas-Acr variant and an sgRNA targeting the AAVS1 (B and C), EMX1 (D and E), and HEK (F and G) locus and incubated for 72 hours followed by T7 endonuclease assay. Representative gel images (B, D, and F) and corresponding quantification of InDel frequencies (C, E, and G). Data are means ± SD; dots are individual data points from n = 3 independent experiments. Ins. 5, insertion variant 5 (see table S2); wt, Cas9 fused to wild-type AcrIIA4.

    Techniques Used: Construct, Variant Assay, Incubation

    Related Articles

    Plasmid Preparation:

    Article Title: Coupling Cas9 to artificial inhibitory domains enhances CRISPR-Cas9 target specificity
    Article Snippet: .. The AcrIIA4 point mutants were created by first amplifying a vector encoding wild-type AcrIIA4 (Addgene no. 113037) with 5′-phosphorylated primers introducing the point mutation(s). .. The resulting vectors were then used as template to generate PCR fragments encoding AcrIIA4 mutants. sgRNA expression vectors were created by inserting target complementary sequences into vector pAAV–RSV–GFP–U6–sgRNA scaffold (Addgene no. 113039) by oligo cloning via Bbs I. BPK4410, a human expression plasmid for SpCas9 Cluster 1 (HypaCas9), was a gift from J. Doudna and K. Joung (Addgene plasmid no. 101178; http://n2t.net/addgene:101178 ; RRID:Addgene_101178). xCas9 3.7 was a gift from D. Liu (Addgene plasmid no. 108379; http://n2t.net/addgene:108379 ; RRID:Addgene_108379). p3s-Sniper-Cas9 was a gift from J. Lee (Addgene plasmid no. 113912; http://n2t.net/addgene:113912 ; RRID:Addgene_113912).

    Mutagenesis:

    Article Title: Coupling Cas9 to artificial inhibitory domains enhances CRISPR-Cas9 target specificity
    Article Snippet: .. The AcrIIA4 point mutants were created by first amplifying a vector encoding wild-type AcrIIA4 (Addgene no. 113037) with 5′-phosphorylated primers introducing the point mutation(s). .. The resulting vectors were then used as template to generate PCR fragments encoding AcrIIA4 mutants. sgRNA expression vectors were created by inserting target complementary sequences into vector pAAV–RSV–GFP–U6–sgRNA scaffold (Addgene no. 113039) by oligo cloning via Bbs I. BPK4410, a human expression plasmid for SpCas9 Cluster 1 (HypaCas9), was a gift from J. Doudna and K. Joung (Addgene plasmid no. 101178; http://n2t.net/addgene:101178 ; RRID:Addgene_101178). xCas9 3.7 was a gift from D. Liu (Addgene plasmid no. 108379; http://n2t.net/addgene:108379 ; RRID:Addgene_108379). p3s-Sniper-Cas9 was a gift from J. Lee (Addgene plasmid no. 113912; http://n2t.net/addgene:113912 ; RRID:Addgene_113912).

    Article Title: Coupling Cas9 to artificial inhibitory domains enhances CRISPR-Cas9 target specificity.
    Article Snippet: .. 2020; 6 : eaay0187 5 February 2020 8 of 11 wild-type AcrIIA4 (Addgene no. 113037) with 5′-phosphorylated primers introducing the point mutation(s). .. The resulting vectors were then used as template to generate PCR fragments encoding AcrIIA4 mutants. sgRNA expression vectors were created by inserting target complementary sequences into vector pAAV–RSV–GFP– U6–sgRNA scaffold (Addgene no. 113039) by oligo cloning via Bbs I. BPK4410, a human expression plasmid for SpCas9 Cluster 1 (HypaCas9), was a gift from J. Doudna and K. Joung (Addgene plasmid no. 101178; http://n2t.net/addgene:101178; RRID:Addgene_101178). xCas9 3.7 was a gift from D. Liu (Addgene plasmid no. 108379; http://n2t.net/addgene:108379; RRID:Addgene_108379). p3s-SniperCas9 was a gift from J. Lee (Addgene plasmid no. 113912; http:// n2t.net/addgene:113912; RRID:Addgene_113912).

    Expressing:

    Article Title: Coupling Cas9 to artificial inhibitory domains enhances CRISPR-Cas9 target specificity.
    Article Snippet: .. Vectors expressing Cas9, Cas9 fused to GFP (Cas9GFP), wild-type AcrIIA4, different AcrIIA4-LOV2 hybrids, or a U6 promoter–driven sgRNA bearing the improved F+E scaffold (38) have been previously reported by us (29, 37) (see Addgene no. 113033- 113039). ..

    Article Title: Coupling Cas9 to artificial inhibitory domains enhances CRISPR-Cas9 target specificity
    Article Snippet: .. Vectors expressing Cas9, Cas9 fused to GFP (Cas9-GFP), wild-type AcrIIA4, different AcrIIA4-LOV2 hybrids, or a U6 promoter–driven sgRNA bearing the improved F+E scaffold ( ) have been previously reported by us ( , ) (see Addgene no. 113033-113039). ..



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    wild type acriia4  (Addgene inc)
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    Addgene inc wild type acriia4
    Fig. 1. Kinetic insulation of CRISPR ON- and OFF-target effects by coexpression of anti-CRISPR proteins. (A) Schematic of a model for Cas9 genome editing. After cotransfec- tion with plasmids encoding Cas9 and sgRNA, plasmids are transcribed to Cas9-mRNA and sgRNA or degraded. Furthermore, the model describes the turnover of mRNAs, sgRNA, Cas9 protein, binding of sgRNA and Cas9, association of Cas9:sgRNA with the target gene, and gene editing. DNAsite, unedited target locus; DNAedited, edited target locus; D:sgR:C, trimeric complex of DNA, sgRNA, and Cas9. (B) Modeling of editing kinetics at high-affinity (ON-target) and low-affinity (OFF-target) sites. Left: The model describes concentrations of the gRNA and Cas9 over time after transient transfection and relates sgRNA and Cas9 expression to a gene-modified fraction of cells. The final gene-edited fraction depends on the integral of Cas9:sgRNA complex expression (upper left panel). Right: Relation between editing efficiency and Cas9 activity (time integral of Cas9:sgRNA complex). The target affinity of an sgRNA determines the editing efficiency at a respective locus. At very large Cas9:sgRNA integrals, gene-edited fractions reach saturation, irrespective of the target affinity. (C) Schematic of constructs used for expression of Cas9, <t>AcrIIA4,</t> and sgRNAs. NLS, nuclear localization signal. (D and E) Coexpressing mild doses of AcrIIA4 improves genome editing specificity. Cells were cotransfected with plasmids encoding AcrIIA4, Cas9, and an sgRNA targeting the AAVS1 locus and incubated for 72 hours followed by T7 endonuclease assay. The AcrIIA4 vector dose used during transfection is indicated. Twenty-two nanograms thereby corresponds to a threefold excess of Cas9/ sgRNA vectors. (D) Representative gel image and (E) quantification of InDel frequencies. (F) HEK 293T cells were cotransduced with 33 l of Cas9 AAV, 33 l of sgRNA AAV, and the indicated volume of AcrIIA4 AAV on two consecutive days. Volumes correspond to the amount of AAV-containing cell lysate applied (see Materials and Methods). Cells were incu- bated for 72 hours followed by T7 endonuclease assay. (E and F) Bars indicate mean editing frequencies; dots are individual data points from n = 3 independent experiments.
    Wild Type Acriia4, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/wild+type+acriia4/AcrIIA4+(Plasmid+%23101043)/pm32076642-146-8-33
    Average 92 stars, based on 1 article reviews
    wild type acriia4 - by Bioz Stars, 2026-09
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    type acriia4  (Addgene inc)
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    Addgene inc type acriia4
    Fig. 1. Kinetic insulation of CRISPR ON- and OFF-target effects by coexpression of anti-CRISPR proteins. (A) Schematic of a model for Cas9 genome editing. After cotransfec- tion with plasmids encoding Cas9 and sgRNA, plasmids are transcribed to Cas9-mRNA and sgRNA or degraded. Furthermore, the model describes the turnover of mRNAs, sgRNA, Cas9 protein, binding of sgRNA and Cas9, association of Cas9:sgRNA with the target gene, and gene editing. DNAsite, unedited target locus; DNAedited, edited target locus; D:sgR:C, trimeric complex of DNA, sgRNA, and Cas9. (B) Modeling of editing kinetics at high-affinity (ON-target) and low-affinity (OFF-target) sites. Left: The model describes concentrations of the gRNA and Cas9 over time after transient transfection and relates sgRNA and Cas9 expression to a gene-modified fraction of cells. The final gene-edited fraction depends on the integral of Cas9:sgRNA complex expression (upper left panel). Right: Relation between editing efficiency and Cas9 activity (time integral of Cas9:sgRNA complex). The target affinity of an sgRNA determines the editing efficiency at a respective locus. At very large Cas9:sgRNA integrals, gene-edited fractions reach saturation, irrespective of the target affinity. (C) Schematic of constructs used for expression of Cas9, <t>AcrIIA4,</t> and sgRNAs. NLS, nuclear localization signal. (D and E) Coexpressing mild doses of AcrIIA4 improves genome editing specificity. Cells were cotransfected with plasmids encoding AcrIIA4, Cas9, and an sgRNA targeting the AAVS1 locus and incubated for 72 hours followed by T7 endonuclease assay. The AcrIIA4 vector dose used during transfection is indicated. Twenty-two nanograms thereby corresponds to a threefold excess of Cas9/ sgRNA vectors. (D) Representative gel image and (E) quantification of InDel frequencies. (F) HEK 293T cells were cotransduced with 33 l of Cas9 AAV, 33 l of sgRNA AAV, and the indicated volume of AcrIIA4 AAV on two consecutive days. Volumes correspond to the amount of AAV-containing cell lysate applied (see Materials and Methods). Cells were incu- bated for 72 hours followed by T7 endonuclease assay. (E and F) Bars indicate mean editing frequencies; dots are individual data points from n = 3 independent experiments.
    Type Acriia4, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/wild+type+acriia4/AcrIIA4+(Plasmid+%23101043)/pm32076642-148-6-32
    Average 92 stars, based on 1 article reviews
    type acriia4 - by Bioz Stars, 2026-09
    92/100 stars
    		  Buy from Supplier

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    Fig. 1. Kinetic insulation of CRISPR ON- and OFF-target effects by coexpression of anti-CRISPR proteins. (A) Schematic of a model for Cas9 genome editing. After cotransfec- tion with plasmids encoding Cas9 and sgRNA, plasmids are transcribed to Cas9-mRNA and sgRNA or degraded. Furthermore, the model describes the turnover of mRNAs, sgRNA, Cas9 protein, binding of sgRNA and Cas9, association of Cas9:sgRNA with the target gene, and gene editing. DNAsite, unedited target locus; DNAedited, edited target locus; D:sgR:C, trimeric complex of DNA, sgRNA, and Cas9. (B) Modeling of editing kinetics at high-affinity (ON-target) and low-affinity (OFF-target) sites. Left: The model describes concentrations of the gRNA and Cas9 over time after transient transfection and relates sgRNA and Cas9 expression to a gene-modified fraction of cells. The final gene-edited fraction depends on the integral of Cas9:sgRNA complex expression (upper left panel). Right: Relation between editing efficiency and Cas9 activity (time integral of Cas9:sgRNA complex). The target affinity of an sgRNA determines the editing efficiency at a respective locus. At very large Cas9:sgRNA integrals, gene-edited fractions reach saturation, irrespective of the target affinity. (C) Schematic of constructs used for expression of Cas9, AcrIIA4, and sgRNAs. NLS, nuclear localization signal. (D and E) Coexpressing mild doses of AcrIIA4 improves genome editing specificity. Cells were cotransfected with plasmids encoding AcrIIA4, Cas9, and an sgRNA targeting the AAVS1 locus and incubated for 72 hours followed by T7 endonuclease assay. The AcrIIA4 vector dose used during transfection is indicated. Twenty-two nanograms thereby corresponds to a threefold excess of Cas9/ sgRNA vectors. (D) Representative gel image and (E) quantification of InDel frequencies. (F) HEK 293T cells were cotransduced with 33 l of Cas9 AAV, 33 l of sgRNA AAV, and the indicated volume of AcrIIA4 AAV on two consecutive days. Volumes correspond to the amount of AAV-containing cell lysate applied (see Materials and Methods). Cells were incu- bated for 72 hours followed by T7 endonuclease assay. (E and F) Bars indicate mean editing frequencies; dots are individual data points from n = 3 independent experiments.

    Journal: Science advances

    Article Title: Coupling Cas9 to artificial inhibitory domains enhances CRISPR-Cas9 target specificity.

    doi: 10.1126/sciadv.aay0187

    Figure Lengend Snippet: Fig. 1. Kinetic insulation of CRISPR ON- and OFF-target effects by coexpression of anti-CRISPR proteins. (A) Schematic of a model for Cas9 genome editing. After cotransfec- tion with plasmids encoding Cas9 and sgRNA, plasmids are transcribed to Cas9-mRNA and sgRNA or degraded. Furthermore, the model describes the turnover of mRNAs, sgRNA, Cas9 protein, binding of sgRNA and Cas9, association of Cas9:sgRNA with the target gene, and gene editing. DNAsite, unedited target locus; DNAedited, edited target locus; D:sgR:C, trimeric complex of DNA, sgRNA, and Cas9. (B) Modeling of editing kinetics at high-affinity (ON-target) and low-affinity (OFF-target) sites. Left: The model describes concentrations of the gRNA and Cas9 over time after transient transfection and relates sgRNA and Cas9 expression to a gene-modified fraction of cells. The final gene-edited fraction depends on the integral of Cas9:sgRNA complex expression (upper left panel). Right: Relation between editing efficiency and Cas9 activity (time integral of Cas9:sgRNA complex). The target affinity of an sgRNA determines the editing efficiency at a respective locus. At very large Cas9:sgRNA integrals, gene-edited fractions reach saturation, irrespective of the target affinity. (C) Schematic of constructs used for expression of Cas9, AcrIIA4, and sgRNAs. NLS, nuclear localization signal. (D and E) Coexpressing mild doses of AcrIIA4 improves genome editing specificity. Cells were cotransfected with plasmids encoding AcrIIA4, Cas9, and an sgRNA targeting the AAVS1 locus and incubated for 72 hours followed by T7 endonuclease assay. The AcrIIA4 vector dose used during transfection is indicated. Twenty-two nanograms thereby corresponds to a threefold excess of Cas9/ sgRNA vectors. (D) Representative gel image and (E) quantification of InDel frequencies. (F) HEK 293T cells were cotransduced with 33 l of Cas9 AAV, 33 l of sgRNA AAV, and the indicated volume of AcrIIA4 AAV on two consecutive days. Volumes correspond to the amount of AAV-containing cell lysate applied (see Materials and Methods). Cells were incu- bated for 72 hours followed by T7 endonuclease assay. (E and F) Bars indicate mean editing frequencies; dots are individual data points from n = 3 independent experiments.

    Article Snippet: Vectors expressing Cas9, Cas9 fused to GFP (Cas9GFP), wild-type AcrIIA4, different AcrIIA4-LOV2 hybrids, or a U6 promoter–driven sgRNA bearing the improved F+E scaffold (38) have been previously reported by us (29, 37) (see Addgene no. 113033- 113039).

    Techniques: Insulation, CRISPR, Protein Binding, Transfection, Expressing, Modification, Activity Assay, Construct, Incubation, Plasmid Preparation

    Fig. 2. Cas-Acr fusion design improves genome editing specificity. (A) Schematic of Cas-Acr constructs comprising Cas9 fused to an artificially weakened AcrIIA4 variant functioning as autoinhibitory domain (AID). (B to G) Cells were cotransfected with plasmids encoding the indicated Cas-Acr variant and an sgRNA targeting the AAVS1 (B and C), EMX1 (D and E), and HEK (F and G) locus and incubated for 72 hours followed by T7 endonuclease assay. Representative gel images (B, D, and F) and corresponding quantification of InDel frequencies (C, E, and G). Data are means ± SD; dots are individual data points from n = 3 independent experiments. Ins. 5, insertion variant 5 (see table S2); wt, Cas9 fused to wild-type AcrIIA4.

    Journal: Science advances

    Article Title: Coupling Cas9 to artificial inhibitory domains enhances CRISPR-Cas9 target specificity.

    doi: 10.1126/sciadv.aay0187

    Figure Lengend Snippet: Fig. 2. Cas-Acr fusion design improves genome editing specificity. (A) Schematic of Cas-Acr constructs comprising Cas9 fused to an artificially weakened AcrIIA4 variant functioning as autoinhibitory domain (AID). (B to G) Cells were cotransfected with plasmids encoding the indicated Cas-Acr variant and an sgRNA targeting the AAVS1 (B and C), EMX1 (D and E), and HEK (F and G) locus and incubated for 72 hours followed by T7 endonuclease assay. Representative gel images (B, D, and F) and corresponding quantification of InDel frequencies (C, E, and G). Data are means ± SD; dots are individual data points from n = 3 independent experiments. Ins. 5, insertion variant 5 (see table S2); wt, Cas9 fused to wild-type AcrIIA4.

    Article Snippet: Vectors expressing Cas9, Cas9 fused to GFP (Cas9GFP), wild-type AcrIIA4, different AcrIIA4-LOV2 hybrids, or a U6 promoter–driven sgRNA bearing the improved F+E scaffold (38) have been previously reported by us (29, 37) (see Addgene no. 113033- 113039).

    Techniques: Construct, Variant Assay, Incubation

    Fig. 1. Kinetic insulation of CRISPR ON- and OFF-target effects by coexpression of anti-CRISPR proteins. (A) Schematic of a model for Cas9 genome editing. After cotransfec- tion with plasmids encoding Cas9 and sgRNA, plasmids are transcribed to Cas9-mRNA and sgRNA or degraded. Furthermore, the model describes the turnover of mRNAs, sgRNA, Cas9 protein, binding of sgRNA and Cas9, association of Cas9:sgRNA with the target gene, and gene editing. DNAsite, unedited target locus; DNAedited, edited target locus; D:sgR:C, trimeric complex of DNA, sgRNA, and Cas9. (B) Modeling of editing kinetics at high-affinity (ON-target) and low-affinity (OFF-target) sites. Left: The model describes concentrations of the gRNA and Cas9 over time after transient transfection and relates sgRNA and Cas9 expression to a gene-modified fraction of cells. The final gene-edited fraction depends on the integral of Cas9:sgRNA complex expression (upper left panel). Right: Relation between editing efficiency and Cas9 activity (time integral of Cas9:sgRNA complex). The target affinity of an sgRNA determines the editing efficiency at a respective locus. At very large Cas9:sgRNA integrals, gene-edited fractions reach saturation, irrespective of the target affinity. (C) Schematic of constructs used for expression of Cas9, AcrIIA4, and sgRNAs. NLS, nuclear localization signal. (D and E) Coexpressing mild doses of AcrIIA4 improves genome editing specificity. Cells were cotransfected with plasmids encoding AcrIIA4, Cas9, and an sgRNA targeting the AAVS1 locus and incubated for 72 hours followed by T7 endonuclease assay. The AcrIIA4 vector dose used during transfection is indicated. Twenty-two nanograms thereby corresponds to a threefold excess of Cas9/ sgRNA vectors. (D) Representative gel image and (E) quantification of InDel frequencies. (F) HEK 293T cells were cotransduced with 33 l of Cas9 AAV, 33 l of sgRNA AAV, and the indicated volume of AcrIIA4 AAV on two consecutive days. Volumes correspond to the amount of AAV-containing cell lysate applied (see Materials and Methods). Cells were incu- bated for 72 hours followed by T7 endonuclease assay. (E and F) Bars indicate mean editing frequencies; dots are individual data points from n = 3 independent experiments.

    Journal: Science advances

    Article Title: Coupling Cas9 to artificial inhibitory domains enhances CRISPR-Cas9 target specificity.

    doi: 10.1126/sciadv.aay0187

    Figure Lengend Snippet: Fig. 1. Kinetic insulation of CRISPR ON- and OFF-target effects by coexpression of anti-CRISPR proteins. (A) Schematic of a model for Cas9 genome editing. After cotransfec- tion with plasmids encoding Cas9 and sgRNA, plasmids are transcribed to Cas9-mRNA and sgRNA or degraded. Furthermore, the model describes the turnover of mRNAs, sgRNA, Cas9 protein, binding of sgRNA and Cas9, association of Cas9:sgRNA with the target gene, and gene editing. DNAsite, unedited target locus; DNAedited, edited target locus; D:sgR:C, trimeric complex of DNA, sgRNA, and Cas9. (B) Modeling of editing kinetics at high-affinity (ON-target) and low-affinity (OFF-target) sites. Left: The model describes concentrations of the gRNA and Cas9 over time after transient transfection and relates sgRNA and Cas9 expression to a gene-modified fraction of cells. The final gene-edited fraction depends on the integral of Cas9:sgRNA complex expression (upper left panel). Right: Relation between editing efficiency and Cas9 activity (time integral of Cas9:sgRNA complex). The target affinity of an sgRNA determines the editing efficiency at a respective locus. At very large Cas9:sgRNA integrals, gene-edited fractions reach saturation, irrespective of the target affinity. (C) Schematic of constructs used for expression of Cas9, AcrIIA4, and sgRNAs. NLS, nuclear localization signal. (D and E) Coexpressing mild doses of AcrIIA4 improves genome editing specificity. Cells were cotransfected with plasmids encoding AcrIIA4, Cas9, and an sgRNA targeting the AAVS1 locus and incubated for 72 hours followed by T7 endonuclease assay. The AcrIIA4 vector dose used during transfection is indicated. Twenty-two nanograms thereby corresponds to a threefold excess of Cas9/ sgRNA vectors. (D) Representative gel image and (E) quantification of InDel frequencies. (F) HEK 293T cells were cotransduced with 33 l of Cas9 AAV, 33 l of sgRNA AAV, and the indicated volume of AcrIIA4 AAV on two consecutive days. Volumes correspond to the amount of AAV-containing cell lysate applied (see Materials and Methods). Cells were incu- bated for 72 hours followed by T7 endonuclease assay. (E and F) Bars indicate mean editing frequencies; dots are individual data points from n = 3 independent experiments.

    Article Snippet: A construct expressing Cas9 fused to wild-type AcrIIA4 via a 40-residue glycine- serine (GS) linker was created by cloning a synthetic DNA fragment encoding the GS linker–AcrIIA4 fragment into vector CMV- SpyCas9 (Addgene no. 103033) via Eco RI/Hind III.

    Techniques: Insulation, CRISPR, Protein Binding, Transfection, Expressing, Modification, Activity Assay, Construct, Incubation, Plasmid Preparation

    Fig. 2. Cas-Acr fusion design improves genome editing specificity. (A) Schematic of Cas-Acr constructs comprising Cas9 fused to an artificially weakened AcrIIA4 variant functioning as autoinhibitory domain (AID). (B to G) Cells were cotransfected with plasmids encoding the indicated Cas-Acr variant and an sgRNA targeting the AAVS1 (B and C), EMX1 (D and E), and HEK (F and G) locus and incubated for 72 hours followed by T7 endonuclease assay. Representative gel images (B, D, and F) and corresponding quantification of InDel frequencies (C, E, and G). Data are means ± SD; dots are individual data points from n = 3 independent experiments. Ins. 5, insertion variant 5 (see table S2); wt, Cas9 fused to wild-type AcrIIA4.

    Journal: Science advances

    Article Title: Coupling Cas9 to artificial inhibitory domains enhances CRISPR-Cas9 target specificity.

    doi: 10.1126/sciadv.aay0187

    Figure Lengend Snippet: Fig. 2. Cas-Acr fusion design improves genome editing specificity. (A) Schematic of Cas-Acr constructs comprising Cas9 fused to an artificially weakened AcrIIA4 variant functioning as autoinhibitory domain (AID). (B to G) Cells were cotransfected with plasmids encoding the indicated Cas-Acr variant and an sgRNA targeting the AAVS1 (B and C), EMX1 (D and E), and HEK (F and G) locus and incubated for 72 hours followed by T7 endonuclease assay. Representative gel images (B, D, and F) and corresponding quantification of InDel frequencies (C, E, and G). Data are means ± SD; dots are individual data points from n = 3 independent experiments. Ins. 5, insertion variant 5 (see table S2); wt, Cas9 fused to wild-type AcrIIA4.

    Article Snippet: A construct expressing Cas9 fused to wild-type AcrIIA4 via a 40-residue glycine- serine (GS) linker was created by cloning a synthetic DNA fragment encoding the GS linker–AcrIIA4 fragment into vector CMV- SpyCas9 (Addgene no. 103033) via Eco RI/Hind III.

    Techniques: Construct, Variant Assay, Incubation