Journal: bioRxiv

Article Title: B cell-specific XIST complex enforces X-inactivation and restrains atypical B cells

doi: 10.1101/2021.01.03.425167

Figure Lengend Snippet: A. RNA-seq analysis showing the XIST RNA expression across control, sgXIST and inhibitors group. B. Heatmap showing Pearson correlation of gene expression allelic bias (d score) on chromosome X between replicates with the control group (Ctrl), XIST perturbation group (sgXIST) and inhibitors treatment group (inhibitors). C. MA plot showing differentially-expressed genes (highlighted in red) between control and inhibitors group (left), or between control and sgXIST group (right). Numbers showing significantly upregulated genes (top) or downregulated genes (down). D. Bar plot showing the percentage of genes on chrX and autosomes between genes that are upregulated in inhibitors group and genes that are upregulated in XIST group compared to control group. P value was calculated using Fisher exact test. E. Box plot showing the log2 counts of expression of genes across the chrX at Xi and Xa in ctrl, sgXIST and inhibitors group. P value was calculated using paired t-test. F. Box plot showing the d score of XIST-dependent and XIST-independent genes in control group using allelic RNA-seq data. P value was calculated using non parametric Mann-Whitney test. G. Bar plot showing the percentage of variable escapee, constitutive escapee and inactive genes between XIST-dependent and -independent genes. P value was calculated using Fisher exact test. H. Gene Ontology analysis showing top GO terms enriched in genes upregulated in sgXIST group (left) or genes upregulated in inhibitors group (right). I. qRT-PCR result of XIST RNA expression of B cells that have been transduced with sgXIST or non-targeting control and then overexpressed with or without anti-CRISPRI protein (ACRIIA4). J. Box plot showing the distribution of d score of allelic gene expression on the X chromosome in groups as in I. P value was calculated using paired t-test. K. Cumulative distribution of d score of allelic gene expression across the X chromosome in groups as in I. P value was calculated using Kolmogorov–Smirnov test.

Article Snippet: To generate the anti-CRISPR virus, we cultured HEK 293T cells at 4 million per 10cm dish and transfected with 4.5 ug pMP.G, 1.5 ug psPAX2 and 6 ug Fuw-AcrIIA4-P2A-GFP vector (Addgene #108247) using OptiMEM and Lipofectamin 3000 at the following day.

Techniques: RNA Sequencing Assay, RNA Expression, Expressing, MANN-WHITNEY, Quantitative RT-PCR, Transduction

Journal: bioRxiv

Article Title: Lack of Cas13a inhibition by anti-CRISPR proteins from Leptotrichia prophages

doi: 10.1101/2021.05.27.445852

Figure Lengend Snippet: ( A, B ) Knockdown of endogenous PPIB ( panel A ) or KRAS ( panel B ) transcripts by LwaCas13a in the presence of various amounts of Acr expression plasmids was determined via RT-qPCR analysis (n = 3 biological replicates; dots represent the mean of three technical triplicate qPCR values with SD shown). PPIB or KRAS RNA levels were normalized to ACTB and knockdown was determined by comparison to a non-targeting LwaCas13a control. Increasing amounts of plasmids encoding the anti-CRISPR proteins AcrVIA5, AcrVIA4, AcrIIA4, and AcrIIA5 were added to a consistent amount of LwaCas13a nuclease and gRNA plasmids. The molar ratios of the Acr to LwaCas13a expression plasmids were approximately 0.12, 0.72, and 4.32 for the 2 ng, 12 ng, and 72 ng treatments, respectively.

Article Snippet: The human codon optimized Acr constructs for AcrVIA4 and AcrVIA5 were synthesized by Twist Biosciences and cloned into a pCMV backbone (plasmid IDs LTH956 and LTH957 respectively; see Supplementary Sequences ), similar to how we previously generated the human cell expression constructs for AcrIIA4 and AcrIIA5 (Addgene IDs 133801 and 133802, respectively) (see Supplementary Sequences ).

Techniques: Knockdown, Expressing, Quantitative RT-PCR, Comparison, Control, CRISPR

Journal: Nucleic acids research

Article Title: Mechanism of Cas9 inhibition by AcrIIA11.

doi: 10.1093/nar/gkaf318

Figure Lengend Snippet: Figure 1. 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 < 0.0 0 01) were determined using a Student’s t -test.

Article Snippet: The sample was placed over a Suerdex 200 Increase 10 / 300 GL (Cytiva, 28990944) equilirated in Fn Cas9 SEC Buffer (200 mM NaCl, 25 mM HEPES, H 7.5, 5% glycerol, and 2 mM DTT), and peak fractions ere collected, spin concentrated, and frozen with liquid ni- rogen before storing at −80 ◦C. uman cell culture genome editing he Sa Cas9 and CMV promoter-driven AcrIIA4 expresion vectors were purchased from Addgene (Plasmid #85452 nd #113038) [ 46 , 47 ].

Techniques: In Vitro, Agarose Gel Electrophoresis, Transfection, Plasmid Preparation, Positive Control, 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: 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: 2020; 6 : eaay0187 5 February 2020 8 of 11 wild-type AcrIIA4 (Addgene no. 113037) with 5′-phosphorylated primers introducing the point mutation(s).

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: 2020; 6 : eaay0187 5 February 2020 8 of 11 wild-type AcrIIA4 (Addgene no. 113037) with 5′-phosphorylated primers introducing the point mutation(s).

Techniques: Construct, Variant Assay, Incubation

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: Frontiers in Bioengineering and Biotechnology

Article Title: GenoMine: a CRISPR-Cas9-based kill switch for biocontainment of Pseudomonas putida

doi: 10.3389/fbioe.2024.1426107

Figure Lengend Snippet: AcrIIA4 as a repressor of ScCas9 in Pseudomonas putida GenoMine (A) Schematic representation of the inhibition design (B) AcrIIA4 does not inhibit ScCas9’s cleavage ability in the GenoMine strain. Targeting efficiency was calculated in Pseudomonas putida GenoMine using the ratio of CFU obtained when transformed with either pSEVAb62-ScCas9 (targeting conditions) or an empty pSEVAb62 (non-targeting conditions), both in the presence and in the absence of a constitutively expressed AcrIIA4 (mean ± s.d., n = 2 biological).

Article Snippet: Third, to measure cleavage survival when the circuits were controlling the expression of AcrIIA4, strains KT2440 and GenoMine were first transformed with the AcrIIA4-containing plasmid pSEVAb23-AcrIIA4.

Techniques: Inhibition, Transformation Assay