Journal: bioRxiv

Article Title: Mechanism of broad-spectrum Cas9 inhibition by AcrIIA11

doi: 10.1101/2021.09.15.460536

Figure Lengend Snippet: (A) HEK293T cells are transiently transfected with plasmids carrying Sa Cas9 + sgRNA. A second plasmid encoding either AcrIIA4 or AcrIIA11 is included, as indicated. (B, C) Representative agarose gel and quantification of indel percentage for the CACNA1D site. Error bars are the standard deviation of three replicates. P-values (not significant [ns], p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001) were determined using a Student’s t-test. (D) Quantification of the editing efficiency when AcrIIA11 or AcrIIA4 are present relative to when no Acr is expressed.

Article Snippet: The Sa Cas9 and CMV promoter-driven AcrIIA4 expression vectors were purchased from Addgene (Plasmid #85452 and #113038) ( , ).

Techniques: Transfection, Plasmid Preparation, Agarose Gel Electrophoresis, Standard Deviation

Journal: Science Advances

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

doi: 10.1126/sciadv.aay0187

Figure Lengend Snippet: ( A ) Schematic of a model for Cas9 genome editing. After cotransfection 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. DNA site , unedited target locus; DNA edited , 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 μl of Cas9 AAV, 33 μl 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 incubated 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: 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).

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

Journal: Science Advances

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

doi: 10.1126/sciadv.aay0187

Figure Lengend Snippet: ( 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: 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).

Techniques: Construct, Variant Assay, Incubation

Journal: Science Advances

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

doi: 10.1126/sciadv.aay0187

Figure Lengend Snippet: ( A ) Overview of the mathematical model of gene editing with Cas-Acr constructs. The model accounts for turnover of plasmids, sgRNA, Cas-Acr mRNA, and protein; transition between the active and inhibited states (Cas-Acr inh ); sgRNA binding (Cas-Acr:sgRNA, Cas-Acr inh :sgRNA); association with a target gene; and gene editing. ( B ) Exemplary model fits to time-resolved T7 endonuclease assay measurements using the AAVS1-targeting sgRNA and either wild-type Cas9 or the Cas-Acr variant Ins. 5 (see fig. S10 for the full set of fits). ( C ) ON- and OFF-target editing efficiencies for sgRNAs targeting the AAVS1, EMX1, RUNX1, or HEK locus are shown together with model simulations of editing efficiencies for either wild-type Cas9 or the indicated Cas-Acr variants. The model was calibrated with ON- and OFF-target editing efficiencies for AAVS1 and ON-target efficiencies for EMX1, RUNX1, and HEK. OFF-target editing measurements for EMX1, RUNX1, and HEK were used for model validation. ( D and E ) Kinetic insulation of ON- and OFF-target editing by Cas-Acr variants. (D) Data points are shown together with inhibitor strengths as estimated by model fitting. Kinetic insulation is achieved for inhibitor strengths that fall between sigmoidal curves for ON- and OFF-target editing. (E) The calibrated model can be used to predict the ratio between ON- and OFF-target editing efficiencies resulting from Cas-Acr variants. The inhibitor strength was defined as the fold change relative to the inhibition rate of Cas-Acr wt, i.e., Cas9 fused to wild-type AcrIIA4. Lines show model simulations; circles indicate measured data points. ( F ) Model simulations of the ratio between ON- and OFF-target editing efficiencies relative to ON-target editing efficiency illustrate the trade-off between Cas9 fidelity and ON-target editing efficiency. Cas-Acr variants can be selected on the basis of highest tolerated OFF-target editing efficiencies.

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).

Techniques: Construct, Binding Assay, Variant Assay, Biomarker Discovery, Insulation, Inhibition

Journal: eLife

Article Title: Functional metagenomics-guided discovery of potent Cas9 inhibitors in the human microbiome

doi: 10.7554/eLife.46540

Figure Lengend Snippet: ( A ) The F01A_2 contig is depicted above the bar chart. Delta symbols (Δ) indicate early stop codons in each gene of the contig. Only the third gene on contig F01A_2 is necessary for SpyCas9 antagonism. ( B ) Induction of the third gene, named acrIIA11 , is sufficient for SpyCas9 antagonism, protecting a plasmid as well as acrIIA4 . Asterisks in ( A ) and ( B ) depict statistically significant differences in plasmid retention between SpyCas9-inducing and non-inducing conditions (Student’s t-test, p<0.01, n = 3); p-values were corrected for multiple hypotheses and ‘ns’ indicates non-significance (p>0.05). Error bars depict standard error of the mean. ( C ) Mu phage fitness, measured by plaquing on E. coli expressing Mu-targeting SpyCas9, is measured in the presence of gfp , acrIIA11 , or acrIIA4 via serial ten-fold dilutions (also see replicated in ). Based on a non-targeting (n.t.) crRNA control, we conclude that SpyCas9 confers ~10 5 fold protection against phage Mu in these conditions. Both acrIIA11 and acrIIA4 significantly enhance Mu fitness by inhibiting SpyCas9.

Article Snippet: The CMV promoter-driven AcrIIA4 expression vector was also purchased from Addgene [Plasmid #113038; ( ).

Techniques: Plasmid Preparation, Expressing

Journal: eLife

Article Title: Functional metagenomics-guided discovery of potent Cas9 inhibitors in the human microbiome

doi: 10.7554/eLife.46540

Figure Lengend Snippet: Mu phage fitness, measured by plaquing on E. coli expressing Mu-targeting SpyCas9, is measured in the presence of gfp , acrIIA11 , or acrIIA4 via serial ten-fold dilutions. Bacterial clearing (black) occurs when phage Mu overcomes SpyCas9 immunity and lyses E. coli . Based on a non-targeting (n.t.) crRNA control, we conclude that SpyCas9 with a targeting (tar) crRNA confers ~10 5 fold protection against phage Mu in these conditions. Both acrIIA11 and acrIIA4 significantly enhance Mu fitness by inhibiting SpyCas9. The indicated anti-CRISPR gene or gfp control is expressed from a second plasmid, in trans.

Article Snippet: The CMV promoter-driven AcrIIA4 expression vector was also purchased from Addgene [Plasmid #113038; ( ).

Techniques: Expressing, CRISPR, Plasmid Preparation

Journal: eLife

Article Title: Functional metagenomics-guided discovery of potent Cas9 inhibitors in the human microbiome

doi: 10.7554/eLife.46540

Figure Lengend Snippet: An EMSA examining the relative mobility of S. pyogenes sgRNA (0.2 µM) through an 8% acrylamide native gel in the presence of SpyCas9 (2 µM) and/or various Acrs (1–32 µM). Neither AcrIIA4 nor AcrIIA11 prevent a gel-shift shift upon SpyCas9 addition, though the nature of the shift is different between Acrs. AcrIIA11 appears to super-shift the SpyCas9/sgRNA complex, which may represent AcrIIA11 bound to this complex. Lanes 10–14 indicate that AcrIIA11 does not readily bind sgRNA. Prominent bands are indicated to the left of the gel and proposed models are cartooned at right. SYBR-Gold was used to visualize sgRNA.

Article Snippet: The CMV promoter-driven AcrIIA4 expression vector was also purchased from Addgene [Plasmid #113038; ( ).

Techniques: Electrophoretic Mobility Shift Assay

Journal: eLife

Article Title: Functional metagenomics-guided discovery of potent Cas9 inhibitors in the human microbiome

doi: 10.7554/eLife.46540

Figure Lengend Snippet: ( A ) AcrIIA11 binds SpyCas9. ( B ) AcrIIA4 binds SpyCas9. SpyCas9 and sgRNA were pre-incubated before mixing with a 2x-strep-tagged AcrIIA11 ( A ) or AcrIIA4 ( B ). SpyCas9 without sgRNA and the meganuclease I-SmaMI were also used. ( A ) Pulldowns on AcrIIA11 brought with them SpyCas9 but not I-SmaMI, and the presence of sgRNA improved the strength of this interaction, but not to the degree seen with AcrIIA4 in ( B ). These images depict total protein content visualized by Coomassie stain. The gel in ( A ) is identical to that depicted in except that the three leftmost control lanes have not been cropped from this image.

Article Snippet: The CMV promoter-driven AcrIIA4 expression vector was also purchased from Addgene [Plasmid #113038; ( ).

Techniques: Incubation, Staining

Journal: eLife

Article Title: Functional metagenomics-guided discovery of potent Cas9 inhibitors in the human microbiome

doi: 10.7554/eLife.46540

Figure Lengend Snippet: ( A ) AcrIIA4 and AcrIIA11a.1 inhibit SpyCas9 cleavage at the CACNA1D locus, as determined via a surveyor nuclease assay with T7 endonuclease I (T7E1). T7E1 cleaves dsDNA that has small insertions and deletions (indels) which result from SpyCas9-induced dsDNA breaks repaired via non-homologous end joining. This allowed for the quantification of SpyCas9 editing efficiency following transient transfection of HEK293T cells. For each experiment, the dagger (†) indicates one of three biological replicates transfected and treated with T7E1 to generate the data in ( B ). ( B ) Quantification of indel frequencies at the CACNA1D locus. Asterisks depict statistically significant differences in indel frequency (Student’s t-Test, n = 3 biological replicates). ( C ) A representative gel image from a single T7E1 assay depicting SpyCas9 cleavage at the EMX1 locus; the dagger (†) indicates samples used to generate the data depicted in ( D ). ( D ) Indel frequencies at the EMX1 locus, as in ( B ). Double asterisks (**), p<0.001; Single asterisk (*), p<0.01; ns, not significant. All p-values were corrected for multiple hypotheses using Bonferroni’s method.

Article Snippet: The CMV promoter-driven AcrIIA4 expression vector was also purchased from Addgene [Plasmid #113038; ( ).

Techniques: Nuclease Assay, Non-Homologous End Joining, Transfection

Journal: eLife

Article Title: Functional metagenomics-guided discovery of potent Cas9 inhibitors in the human microbiome

doi: 10.7554/eLife.46540

Figure Lengend Snippet: ( A ) Quantification of indel frequencies at the CACNA1D and EMX1 loci after transient plasmid transfection of human HEK293T cells. Asterisks depict statistically significant differences in indel frequency (Student’s t-Test, n = 3 biological replicates). Double asterisks (**), p<0.001; ns, not significant. All p-values were corrected for multiple hypotheses using Bonferroni’s method. ( B ) Western blot on transfected HEK293T cells shows that AcrIIA11a.1 and AcrIIA11b.1 express comparably well, independent of SpyCas9 co-transfection. Additionally, SpyCas9 is expressed to similar levels with all Acrs tested. AcrIIA4 was not HA-tagged, so no signal is seen for this Acr.

Article Snippet: The CMV promoter-driven AcrIIA4 expression vector was also purchased from Addgene [Plasmid #113038; ( ).

Techniques: Plasmid Preparation, Transfection, Western Blot, Cotransfection

Journal: bioRxiv

Article Title: CRISPR-mediated biocontainment

doi: 10.1101/2020.02.03.922146

Figure Lengend Snippet: ( a ) Prevention of DNA edits in HEK 293T cells at the JAK2 locus by sgRNA-Alu, anti-CRISPR protein AcrIIa4, and sgRNA targeting essential gene POLE2. The percentage of reads with an indel is plotted on the y-axis and represents the mean of three biological replicates. ( b ) Three days after transfection, apoptosis was measured by FACS using Annexin V and propidium iodide staining in HEK 293T cells transiently expressing Cas9 and sgRNA-Alu, AcrIIa4, or sgRNA-POLE2. The FACS plots showing Annexin V on the x-axis and propidium iodide on the y-axis in HEK 293T are displayed for each sample. ( c ) Percentage of apoptotic cells is plotted on the y-axis with early apoptosis in light blue as measured by Annexin V+ propidium iodide-, and late apoptosis in light orange as measured by Annexin V+ Propidium iodide+ FACS populations. In all histograms, error bars represent standard error, n=3. Student t-tests were performed and marked NS, not significant (P > 0.05); *P < 0.05; **P < 0.01; ***P < 0.001 as compared to the positive control in (a) and to the negative control in (c).

Article Snippet: Expression vectors encoding Anti-CRISPR proteins were obtained from Addgene: AcrIIA2 (pJH373 plasmid, ID# 86840) and AcrIIA4 (pJH376 plasmid, ID# 86840).

Techniques: CRISPR, Transfection, Staining, Expressing, Positive Control, Negative Control

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

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

Journal: Science Advances

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

doi: 10.1126/sciadv.aay0187

Figure Lengend Snippet: ( A ) Schematic of a model for Cas9 genome editing. After cotransfection 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. DNA site , unedited target locus; DNA edited , 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 μl of Cas9 AAV, 33 μl 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 incubated 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- Spy Cas9 (Addgene no. 103033) via Eco RI/Hind III.

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

Journal: Science Advances

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

doi: 10.1126/sciadv.aay0187

Figure Lengend Snippet: ( 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- Spy Cas9 (Addgene no. 103033) via Eco RI/Hind III.

Techniques: Construct, Variant Assay, Incubation

Journal: Science Advances

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

doi: 10.1126/sciadv.aay0187

Figure Lengend Snippet: Cells were cotransfected with plasmids encoding the indicated Cas9 variant and an sgRNA targeting the AAVS1, RUNX, HBB, or EMX1 locus. Following incubation for 72 hours, a T7 endonuclease assay was performed. Data are means ± SD; 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- Spy Cas9 (Addgene no. 103033) via Eco RI/Hind III.

Techniques: Variant Assay, Incubation

Journal: Science Advances

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

doi: 10.1126/sciadv.aay0187

Figure Lengend Snippet: ( A ) Overview of the mathematical model of gene editing with Cas-Acr constructs. The model accounts for turnover of plasmids, sgRNA, Cas-Acr mRNA, and protein; transition between the active and inhibited states (Cas-Acr inh ); sgRNA binding (Cas-Acr:sgRNA, Cas-Acr inh :sgRNA); association with a target gene; and gene editing. ( B ) Exemplary model fits to time-resolved T7 endonuclease assay measurements using the AAVS1-targeting sgRNA and either wild-type Cas9 or the Cas-Acr variant Ins. 5 (see fig. S10 for the full set of fits). ( C ) ON- and OFF-target editing efficiencies for sgRNAs targeting the AAVS1, EMX1, RUNX1, or HEK locus are shown together with model simulations of editing efficiencies for either wild-type Cas9 or the indicated Cas-Acr variants. The model was calibrated with ON- and OFF-target editing efficiencies for AAVS1 and ON-target efficiencies for EMX1, RUNX1, and HEK. OFF-target editing measurements for EMX1, RUNX1, and HEK were used for model validation. ( D and E ) Kinetic insulation of ON- and OFF-target editing by Cas-Acr variants. (D) Data points are shown together with inhibitor strengths as estimated by model fitting. Kinetic insulation is achieved for inhibitor strengths that fall between sigmoidal curves for ON- and OFF-target editing. (E) The calibrated model can be used to predict the ratio between ON- and OFF-target editing efficiencies resulting from Cas-Acr variants. The inhibitor strength was defined as the fold change relative to the inhibition rate of Cas-Acr wt, i.e., Cas9 fused to wild-type AcrIIA4. Lines show model simulations; circles indicate measured data points. ( F ) Model simulations of the ratio between ON- and OFF-target editing efficiencies relative to ON-target editing efficiency illustrate the trade-off between Cas9 fidelity and ON-target editing efficiency. Cas-Acr variants can be selected on the basis of highest tolerated OFF-target editing efficiencies.

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- Spy Cas9 (Addgene no. 103033) via Eco RI/Hind III.

Techniques: Construct, Binding Assay, Variant Assay, Biomarker Discovery, Insulation, Inhibition

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: 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).

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

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: 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).

Techniques: Construct, Variant Assay, Incubation