cmv promoter driven acriia4 expression vectors  (Addgene inc)

Journal: Nucleic Acids Research

Article Title: Mechanism of Cas9 inhibition by AcrIIA11

doi: 10.1093/nar/gkaf318

Figure Lengend Snippet: AcrIIA11 inhibits SaCas9 in vitro and in human HEK293T cells. ( A ) Agarose gel and ( B ) quantification of DNA cleavage by SaCas9. Graphs represent the mean of three replicates. Error bars: S.E.M. ( C ) HEK293Ts are transiently transfected with plasmids carrying SaCas9 + sgRNA. A second plasmid encodes either AcrIIA4 (positive control) or AcrIIA11. ( D ) Representative agarose gel and ( E ) 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 < 0.001; **** P < 0.0001) were determined using a Student’s t -test. ( F ) Quantification of the editing efficiency when AcrIIA11 or AcrIIA4 are present relative to when no Acr is expressed. Error bars are the standard deviation of three replicates. P -values (not significant [ns], P > 0.05; **** P < 0.0001) were determined using a Student’s t -test.

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

Techniques: In Vitro, Agarose Gel Electrophoresis, Transfection, Plasmid Preparation, Positive Control, Standard Deviation

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: 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