Journal: Nucleic Acids Research

Article Title: Small-molecule activators specific to adenine base editors through blocking the canonical TGF-β pathway

doi: 10.1093/nar/gkac742

Figure Lengend Snippet: Effects of SB505124 on SpCas9 and CBEs. ( A–D ) Effect of SB505124 on SpCas9. Heat maps showing the genome cutting efficiencies at five indicated sites treated by SB505124 or DMSO ( n = 3) (A). The average mutation rates for all sites tested are quantified and compared with the DMSO group (B). The indel rates quantified using NGS for all individual sites tested (C) and statistical analysis for all sites (D) are calculated and compared with the DMSO group. ( E ) Sanger sequencing results showing the editing efficiencies of BE3 at 10 positions at six genomic loci. ( F ) Sanger sequencing results showing the editing efficiencies of four indicated CBE variants at the EMX1 and RNF2 loci. ( G ) Statistical analysis of the normalized editing frequencies of BE3 for all six sites tested in (E). ( H ) Statistical analysis of the normalized editing frequencies of four indicated CBE variants at EMX1 and RNF2 as shown in (F). ( I ). Bar graphs showing the NGS results of the indel frequencies induced by BE3, as quantified at six indicated genome sites. ( J ) Fraction of on-target cytosine substitutions for BE3 at six indicated sites as analyzed using NGS. Data are presented as the mean ± SD and are the results of three technical replicates. P -values were determined using a two-tailed Student's t -test.

Article Snippet: Membranes were blocked in 5% non-fat dry milk in Tris-buffered saline with Tween-20 (TBST) for at least 1 h at room temperature followed by incubating with anti-SpCas9 antibody (CST, Cat. No. 14697T, 1:1000) diluted in TBST overnight at 4°C.

Techniques: Mutagenesis, Sequencing, Two Tailed Test

Journal: Molecular Therapy. Nucleic Acids

Article Title: Inhibition of adenovirus replication by CRISPR-Cas9-mediated targeting of the viral E1A gene

doi: 10.1016/j.omtn.2023.02.033

Figure Lengend Snippet: Structure of the recombinant adenovirus vector constructs and gRNA sequences incorporated into them (A) Schematic of HAdV-5-based vectors with inserted individual gRNA sequences. All adenoviral vectors are based on the HAdV-5-derived vector pAd/PL-DEST (Thermo Fisher Scientific) and lack the E1 and E3 regions. Cas9 and gRNA expression cassettes were inserted into the deleted E1 region. The expression of spCas9-HF1 is driven by a tetracycline repressor-controlled CMV promoter comprising two binding sites for the repressor (2×TetO2). The expression of the individual targeting or non-targeting gRNAs is under control of a constitutive human U6 (hU6) promoter. The structure and sequence of the gRNAs are exemplarily shown for E1A gRNA 9 bound to its target site. The control vector containing only the Cas9 expression cassette is also depicted. (B) Target sequences for the individual gRNAs and their positions within the HAdV-5 genome (AY339865.1).

Article Snippet: SpCas9-HF1 and β-actin were detected with antibodies 7A9-3A3 (Cell Signaling Technology, Danvers, MA, USA) and GTX629630-25 (GeneTex, Irvine, CA, USA), respectively.

Techniques: Recombinant, Plasmid Preparation, Construct, Derivative Assay, Expressing, Binding Assay, Sequencing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Inhibition of adenovirus replication by CRISPR-Cas9-mediated targeting of the viral E1A gene

doi: 10.1016/j.omtn.2023.02.033

Figure Lengend Snippet: Targeting E1A with 4 different gRNAs potently inhibits HAdV-5 replication (A) Schematic of the HAdV-5-based CRISPR-Cas9 vectors for multiplex gRNA expression. The sequences of gRNAs 1, 7, 8, and 9 were expressed from individual constitutive RNA polymerase III promoters (mU6, h7SK, hH1, and hU6). An analogous vector containing 4 identical NT gRNAs instead of the targeting gRNAs was constructed as well. SpCas9-HF1 was expressed from a doxycycline-regulatable CMV promoter harboring two TetO2 binding sites for the tetracycline repressor. (B) HeLa cells were transduced with the adenoviral vector containing Cas9 in combination with gRNAs 1, 7, 8, and 9 or with a control vector carrying 4 NTNT gRNAs at an MOI of 100, followed by infection of the cells with HAdV-5 at an MOI of 0.01 24 h later. Numbers of infectious virus particles were determined at the indicated time points and expressed as IFUs per milliliter. Data represent the means (n = 3) ± SD of triplicate infections of a representative experiment of 3. ∗p < 0.05, ∗∗∗p < 0.001. (C) Experimental settings were as in (A) with the difference that HeLa cells were infected with HAdV-5 6 h prior to transduction with the vectors. Numbers of infectious virus particles were determined on day 2 post infection and expressed as IFUs per milliliter. Data represent the means (n = 3) ± SD of triplicate infections of a representative experiment of 3. ∗∗p < 0.01.

Article Snippet: SpCas9-HF1 and β-actin were detected with antibodies 7A9-3A3 (Cell Signaling Technology, Danvers, MA, USA) and GTX629630-25 (GeneTex, Irvine, CA, USA), respectively.

Techniques: CRISPR, Multiplex Assay, Expressing, Plasmid Preparation, Construct, Binding Assay, Transduction, Infection

Journal: Molecular Therapy. Nucleic Acids

Article Title: Inhibition of adenovirus replication by CRISPR-Cas9-mediated targeting of the viral E1A gene

doi: 10.1016/j.omtn.2023.02.033

Figure Lengend Snippet: Concatemerized gRNAs separated by NT amiRNA spacers allow potent inhibition of HAdV-5 replication (A) Schematic of the adenoviral vector for multiplex expression of concatemerized gRNAs and processing of the primary transcripts within the cell. SpCas9-HF1 expression is under control of a doxycycline-regulatable CMV promoter harboring two TetO2 binding sites for the tetracycline repressor, and multiplexed gRNAs separated by NT amiRNA are expressed from a common hU6 promoter. A negative control vector carrying 4 NT gRNAs was constructed in an analogous manner. The individual gRNAs are released from the primary transcript by DROSHA-mediated cleavage at the base of each interjacent amiRNA hairpin. The gRNA intermediates are further trimmed by Cas9 to give rise to the mature gRNAs. (B) HeLa cells were transduced with the adenoviral vectors containing Cas9 in combination with the concatemerized targeting gRNAs 1, 7, 8, and 9 or with a control vector bearing Cas9 in combination with 4 NT gRNAs at an MOI of 100. 24 h after transduction, the cells were infected with HAdV-5 at an MOI of 0.01. Numbers of infectious virus particles were determined at the indicated time points and expressed as IFUs per milliliter. For better comparison, the values for the vectors expressing the 4 gRNAs from individual promoters and those for the only-HAdV-5 control presented in Figure 6 D are also shown here because the respective transfection/infection experiments were conducted side by side. Data represent the means (n = 3) ± SD of triplicate infections of a representative experiment of 3. ∗p < 0.05, ∗∗∗p < 0.001. (C) Experimental settings were as in (B) with the difference that HeLa cells were infected with HAdV-5 6 h prior to transduction with the vectors. Numbers of infectious virus particles were determined on day 2 post infection and expressed as IFUs per milliliter. Data represent the means (n = 3) ± SD of triplicate infections of a representative experiment of 3. ∗p < 0.05.

Article Snippet: SpCas9-HF1 and β-actin were detected with antibodies 7A9-3A3 (Cell Signaling Technology, Danvers, MA, USA) and GTX629630-25 (GeneTex, Irvine, CA, USA), respectively.

Techniques: Inhibition, Plasmid Preparation, Multiplex Assay, Expressing, Binding Assay, Negative Control, Construct, Transduction, Infection, Transfection

Journal: PLOS ONE

Article Title: Small-molecule inhibitors of proteasome increase CjCas9 protein stability

doi: 10.1371/journal.pone.0280353

Figure Lengend Snippet: A ) Schematic representation of the size of SpCas9 and CjCas9. B) The HEK293T cells were transfected with the same number of copies of plasmids of either pX551-CMV-SpCas9 or of pX551-CMV-CjCas9. 72 h later, the numbers of plasmids inside the cells were determined by qPCR. C ) CjCas9 and SpCas9 mRNAs were assessed by RT-qPCR with HPRT as the reference gene. D ) The concentrations of the two Cas9 proteins fused with HA-tag were evaluated by western blot. Data are means ± SEM (n ≥ 3), *p < 0.05, **p < 0.005, and ***p < 0.0005 (Student’s t tests).

Article Snippet: The CjCas9 and SpCas9 protein fused with HA-tag were detected using anti-HA tag (1:1,000; Cell Signaling Technology, Danvers, MA) and β-actin with anti-β-actin (1:1,000; Cell Signaling Technology) antibodies, respectively, and visualized with HRP conjugated anti-rabbit secondary antibody (1:20,000; Dianova, Hamburg, Germany) using West Pico Chemiluminescence substrate (Thermo Fisher Scientific, Waltham, MA).

Techniques: Transfection, Quantitative RT-PCR, Western Blot

Journal: ACS Nano

Article Title: Efficient Genome Editing Achieved via Plug-and-Play Adenovirus Piggyback Transport of Cas9/gRNA Complex on Viral Capsid Surface

doi: 10.1021/acsnano.2c00909

Figure Lengend Snippet: Cas9SpC retained CRISPR nuclease activity. (A) Cas9 and Cas9SpC with the indicated amounts were resolved on an SDS-PAGE gel followed by Coomassie blue staining (left) or by immunoblotting with anti-SpCas9 and anti-6xHis antibodies (middle and right). (B) Schematic diagram of the Rosa26:Ai9-SauSpyCas9-tdTomato (mAi9) locus without or with gene editing. Three SV40 polyadenylation signals (STOP) function as a potent transcription inhibition element to the downstream tdTomato gene. Endonuclease cleavage at both upstream and downstream sites by Cas9/lox gRNA can lead to a subset of alleles with deletion of the STOP cassette and activation of the tdTomato gene expression. The locations of duplicate lox gRNA sites and PCR genotyping primers are illustrated. (C and D) Scatter plots with a bar graph showing the percentage of tdTomato-positive cells by fluorescence microscopy analysis of mAi9 cells on day 4 following lipofectamine CRISPRMAX transfection with Cas9/lox gRNA or with Cas9SpC/lox gRNA. The numbers below the graphs indicate the amount of reagents in picomoles added to 1 × 10 5 mAi9 cells in 500 μL of culture media in a 24-well format. (E) Top: Flow cytometry analysis of the percentage of tdTomato-positive mAi9 cells on day 4 following transfection with 7.5 pmol of Cas9/lox gRNA or with 7.5 pmol of Cas9SpC/lox gRNA to 5 × 10 4 mAi9 cells in 500 μL of culture media in a 24-well format. Cells a receiving mock treatment with phosphate-buffered saline were used to set the gate for detection of tdTomato-positive cells. Bottom: Scatter plot with a bar graph showing flow cytometry analysis of the percentage of tdTomato-positive cells on day 4 following transfection with Cas9/lox gRNA or with Cas9SpC/lox gRNA to mAi9 cells seeded at varying seeding cell densities in a 24-well format. Two replicate experiments were performed for parts C–E. Data are represented as mean ± standard deviation from six wells of two replicate experiments for parts C and D and as three wells of one representative experiment for part E.

Article Snippet: Primary antibodies used in this study included mouse anti-SpCas9 (Cell Signaling Technology, #14697, 1:400) and goat anti-Ad5 hexon.

Techniques: CRISPR, Activity Assay, SDS Page, Staining, Western Blot, Inhibition, Activation Assay, Expressing, Fluorescence, Microscopy, Transfection, Flow Cytometry, Standard Deviation

Journal: ACS Nano

Article Title: Efficient Genome Editing Achieved via Plug-and-Play Adenovirus Piggyback Transport of Cas9/gRNA Complex on Viral Capsid Surface

doi: 10.1021/acsnano.2c00909

Figure Lengend Snippet: Cas9SpC retained CRISPR nuclease activity. (A) Cas9 and Cas9SpC with the indicated amounts were resolved on an SDS-PAGE gel followed by Coomassie blue staining (left) or by immunoblotting with anti-SpCas9 and anti-6xHis antibodies (middle and right). (B) Schematic diagram of the Rosa26:Ai9-SauSpyCas9-tdTomato (mAi9) locus without or with gene editing. Three SV40 polyadenylation signals (STOP) function as a potent transcription inhibition element to the downstream tdTomato gene. Endonuclease cleavage at both upstream and downstream sites by Cas9/lox gRNA can lead to a subset of alleles with deletion of the STOP cassette and activation of the tdTomato gene expression. The locations of duplicate lox gRNA sites and PCR genotyping primers are illustrated. (C and D) Scatter plots with a bar graph showing the percentage of tdTomato-positive cells by fluorescence microscopy analysis of mAi9 cells on day 4 following lipofectamine CRISPRMAX transfection with Cas9/lox gRNA or with Cas9SpC/lox gRNA. The numbers below the graphs indicate the amount of reagents in picomoles added to 1 × 10 5 mAi9 cells in 500 μL of culture media in a 24-well format. (E) Top: Flow cytometry analysis of the percentage of tdTomato-positive mAi9 cells on day 4 following transfection with 7.5 pmol of Cas9/lox gRNA or with 7.5 pmol of Cas9SpC/lox gRNA to 5 × 10 4 mAi9 cells in 500 μL of culture media in a 24-well format. Cells a receiving mock treatment with phosphate-buffered saline were used to set the gate for detection of tdTomato-positive cells. Bottom: Scatter plot with a bar graph showing flow cytometry analysis of the percentage of tdTomato-positive cells on day 4 following transfection with Cas9/lox gRNA or with Cas9SpC/lox gRNA to mAi9 cells seeded at varying seeding cell densities in a 24-well format. Two replicate experiments were performed for parts C–E. Data are represented as mean ± standard deviation from six wells of two replicate experiments for parts C and D and as three wells of one representative experiment for part E.

Article Snippet: Membranes were blocked in Tris-buffered saline (TBS, pH 7.6) containing 0.5% Tween 20 (TBST) and 5% nonfat dry milk and incubated in 5% BSA in TBST, containing rabbit polyclonal anti-SpCas9 (Cell Signaling Technology, #14697, 1:5,000) and mouse monoclonal anti-6xHis (MilliporeSigma, H1029, 1:1,000) antibodies overnight.

Techniques: CRISPR, Activity Assay, SDS Page, Staining, Western Blot, Inhibition, Activation Assay, Expressing, Fluorescence, Microscopy, Transfection, Flow Cytometry, Standard Deviation

Journal: eLife

Article Title: An optimized CRISPR/Cas9 approach for precise genome editing in neurons

doi: 10.7554/eLife.65202

Figure Lengend Snippet: ( A ) Schematic of in utero electroporation and ( B ) subsequent in vivo two-photon microscopy setup. ( C ) Two-photon 3D reconstruction of a layer 2/3 pyramidal neuron with TKIT-mediated SEP-GluA2 KI. ( D ) Maximum intensity projection of the same example cell in ( C ) with magnified dendritic segments (white boxes). Scale bars are shown in the bottom right corner of each image. ( E ) Dual AAV-HA-SpCas9 and AAV-TKIT-SEP-GluA2 injections were made into the dorsal hippocampus of adult C57BL/6N mice. ( F ) Confocal image of native SEP-GluA2 fluorescence signal. The CA1 cell layer is illustrated with a magenta dotted line with dendrites in Stratum radiatum. The image is a maximum intensity projection. ( G ) A composite confocal image showing the expression of SEP-GluA2 (GFP antibody, green), Cas9 (HA antibody, magenta), and nuclei (DAPI, blue). The image is a maximum intensity projection and is centered on the CA1 pyramidal cell layer. ( H ) Quantification of the 16.1% of HA-SpCas9 expressing cells that are also GFP positive. Scale bars are indicated in the bottom right corner of images. Figure 5—source data 1. Immunohistochemistry data for SEP-GluA2 knockin efficiency calculation with dual virus approach.

Article Snippet: For detection of AAV-mediated spCas9 expression the same immunohistochemistry protocol was followed but with the addition of an antibody against HA (Cell Signaling, Rabbit C29F4, 1:500).

Techniques: In Utero, Electroporation, In Vivo, Microscopy, Fluorescence, Expressing, Immunohistochemistry, Knock-In

Journal: eLife

Article Title: An optimized CRISPR/Cas9 approach for precise genome editing in neurons

doi: 10.7554/eLife.65202

Figure Lengend Snippet:

Article Snippet: For detection of AAV-mediated spCas9 expression the same immunohistochemistry protocol was followed but with the addition of an antibody against HA (Cell Signaling, Rabbit C29F4, 1:500).

Techniques: Plasmid Preparation, In Vivo, Isolation, Cell Culture, Recombinant, Clone Assay, Software, CRISPR