Journal: Scientific Reports

Article Title: Rosa26-LSL-dCas9-VPR: a versatile mouse model for tissue specific and simultaneous activation of multiple genes for drug discovery

doi: 10.1038/s41598-022-23127-7

Figure Lengend Snippet: Generation and characterization of Cre-dependent dCas9-VPR-expressing mice. ( A ) Scheme of the Cre-dependent dCas9-VPR Rosa26 targeting vector. ( B ) Outline of the in vivo experiment. Rosa26-LSL-dCas9-VPR mice were iv injected with AAV8 containing LP1-Cre gene (AAV8- Cre ) or 6 different gRNAs against Pcsk9 (AAV8- gPcsk9 ), named gPcsk9-1 to 6 , at the amount of 1 × 10 11 VG/mouse. 21 days later mice were sacrificed, and tissues were collected for analysis. ( C ) PCR representation showing LSL cassette recombination in liver tissues isolated from Rosa26-LSL-dCas9-VPR mice transduced with AAV8- Cre or AAV8- gPCSK9 alone. A representative agarose gel electrophoresis image is shown. Lane 1–3 contains amplicons obtained from tissue samples of three different mice treated with AAV8- gPcsk9 and lanes 4–6 from tissue samples of three different mice treated with AAV8- Cre . The expected size of PCR products, marker (M) and NTC (H2O) are indicated. ( D ) dCas9-VPR RNA expression in tissues dissected from AAV8- Cre or AAV8- gPCSK9 injected Rosa26-LSL-dCas9-VPR mice. dCas9-VPR expression can only be seen in AAV8- Cre treated Rosa26-LSL-dCas9-VPR mice liver samples. Mean values are shown as a relative quantification, with corresponding expression level of AAV- Cre treated control group as a reference (n = 10).

Article Snippet: LSL cassette recombination PCR was performed on liver cDNA using Quick-Load® Taq Master Mix (M0271S, NEB) with primers p1, p3, p2 (Supplementary Table ).

Techniques: Expressing, Plasmid Preparation, In Vivo, Injection, Isolation, Transduction, Agarose Gel Electrophoresis, Marker, RNA Expression, Quantitative Proteomics, Control

Journal: Nature Genetics

Article Title: Deep CRISPR mutagenesis characterizes the functional diversity of TP53 mutations

doi: 10.1038/s41588-024-02039-4

Figure Lengend Snippet: a , Scheme for CRISPR–Cas9-mediated TP53 mutagenesis via homology-directed repair (HDR) in HCT116 LSL/Δ cell line. b , Editing efficiency as percentage of single-cell clones that contain a targeted integration of the donor and the desired mutation analyzed by PCR and sequencing, respectively. Shown are results for single mutations and the mean across the panel. c , Western blot demonstrating mutant p53 and p21 protein expression in HCT116 clones after Cre-mediated excision of the LSL cassette in absence and presence of 10 µM N3a. d , Principal component analysis based on RNA-seq data of indicated cell clones ±N3a. e , Gene set enrichment analysis for p53-related gene expression signatures comparing indicated N3a- and DMSO-treated cell clones. f , g , Proliferation of TP53 -mutant cell clones in presence of increasing concentrations of N3a analyzed by real-time live-cell imaging. f , Area under the proliferation curve relative to untreated. g , 50% inhibitory concentration (IC 50 , with 95% CI) for N3a with p53-null (LSL, red) and WT (green) as reference. 95% CI, 95% confidence interval; AUC, area under the curve; FDRq, false discovery rate q-value; LSL, LoxP-Stop-LoxP; Puro, puromycin.

Article Snippet: First, 2.5 × 10 4 HCT116 cells were transfected with 1.25 µg of pX330_sgIn5 (sgRNA 5′-TCA GTG AGG AAT CAG AGG CC-3′) and 1.25 µg of HR700, which contained WT homology arms 1 and 2 flanking the LSL cassette, using Lipofectamine 2000 (Thermo Fisher Scientific, cat. no. 11668019) according to the manufacturer’s protocol.

Techniques: CRISPR, Mutagenesis, Clone Assay, Sequencing, Western Blot, Expressing, RNA Sequencing Assay, Live Cell Imaging, Concentration Assay

Journal: Nature Genetics

Article Title: Deep CRISPR mutagenesis characterizes the functional diversity of TP53 mutations

doi: 10.1038/s41588-024-02039-4

Figure Lengend Snippet: a , CRISPR/Cas9-targeting of TP53 in HCT116 cells. Shown are the two TP53 alleles and their modifications. The Δ allele contains inactivating intronic deletions ( b and c ). The second allele contains a loxP-flanked transcriptional stop (LSL) cassette, expressing GFP and a non-functional puromycin N-acetyltransferase (Puro mut ) resistance gene, and harbors an SNV for allele-specific Cas9-targeting. Donor vectors contain an intact puromycin resistance gene allowing selection of HDR-edited cells. To prevent re-cutting and enable selective amplification of edited alleles (LSL-mut), exon 5/6 donors contained a PAM-inactivating mutation. An intron-7 deletion on the LSL allele eliminated the need for an additional exon 7/8 donor mutation. Adenoviral Cre was used to excise the LSL-cassette and activate expression, yielding HCT116 mut/Δ cells. Selective NGS of the edited and Cre-recombined allele was ensured by nested PCR using the indicated primer pairs. d and e , Western blot of p53 and p21 expression in the indicated cell lines treated with Cre and N3a as indicated. f and g , Proliferation of TP53 -mutant cell clones in the presence of N3a analyzed by real-time live-cell imaging. Shown is the area under the proliferation curve (AUC) relative to untreated. p53-null (LSL, red) and wild type (WT, green) are shown for reference. h-j , p53 protein expression in edited HCT116 and H460 cells, normal human diploid fibroblasts (NHDF, two donors), mammary epithelial cells (MCF10A), normal human epidermal keratinocytes (NHEK), and patient-derived p53-mutant cell lines. j , Quantification of p53 normalized to β-actin in ( i ). Mean±SD (n=5 cell lines per group); one-way ANOVA with Holm-Šídák's multiple comparisons test.

Article Snippet: First, 2.5 × 10 4 HCT116 cells were transfected with 1.25 µg of pX330_sgIn5 (sgRNA 5′-TCA GTG AGG AAT CAG AGG CC-3′) and 1.25 µg of HR700, which contained WT homology arms 1 and 2 flanking the LSL cassette, using Lipofectamine 2000 (Thermo Fisher Scientific, cat. no. 11668019) according to the manufacturer’s protocol.

Techniques: CRISPR, Expressing, Functional Assay, Selection, Amplification, Mutagenesis, Nested PCR, Western Blot, Clone Assay, Live Cell Imaging, Derivative Assay

Journal: Nature Genetics

Article Title: Deep CRISPR mutagenesis characterizes the functional diversity of TP53 mutations

doi: 10.1038/s41588-024-02039-4

Figure Lengend Snippet: a , b , Comparison of different stress factors. a , Heatmap showing changes in variant abundance in response to DNA damage (IR, ionizing radiation; 5-FU, 5-fluorouracil) or nutrient starvation (−Glc, glucose starvation; −Gln, glutamine starvation) compared with control treatment with DMSO and N3a. Shown is the enrichment as the −log 2 fold abundance change relative to the mean of the controls: unirradiated cells for IR samples, untreated cells for 5-FU samples and unstarved cells in regular growth medium for starvation samples ( n = 3 biological replicates per condition). b , Scatter plots illustrating the correlation between enrichment under DNA damage or nutrient deprivation and specific p53 activation with N3a. Shown is the mean ± s.d. enrichment ( n = 3 biological replicates). Dashed line, line of identity. c – e , Proapoptotic activity of R175 variants. c , Experimental scheme and a representative FACS scatter plot demonstrating the sorting strategy based on annexin V staining. GFP-negative (neg) cells were gated to selectively analyze cells expressing the p53 variant, that is, cells with successful deletion of the GFP-expressing LSL cassette after AV-Cre infection. d , Heatmap illustrating N3a-induced changes in variant abundance in the annexin V-positive (pos) fraction (left) compared with the entire cell pool (right). Shown is the −log 2 fold change ( n = 3 biological replicates) relative to the annexin V-negative fraction (left) or DMSO-treated control cells (right). Lanes labeled as ‘16 d–4 d’ represent the difference between the 4 d and 16 d timepoint, reflecting late N3a-induced changes in variant abundance. e , Scatter plot showing the correlation between the early (4 d) and late (between 4 and 16 d) occurring N3a-induced changes in variant abundance versus their enrichment in the apoptotic cell fraction. Shown is the mean ± s.d. enrichment ( n = 3 biological replicates) relative to the DMSO-treated control. All scatter plots show the Pearson correlation coefficient ρ with P value approximated using a two-tailed t -distribution and kernel density estimation plots on the side to illustrate the separation of variant classes. Dashed line, line of identity. FACS, fluorescence-activated cell sorting.

Article Snippet: First, 2.5 × 10 4 HCT116 cells were transfected with 1.25 µg of pX330_sgIn5 (sgRNA 5′-TCA GTG AGG AAT CAG AGG CC-3′) and 1.25 µg of HR700, which contained WT homology arms 1 and 2 flanking the LSL cassette, using Lipofectamine 2000 (Thermo Fisher Scientific, cat. no. 11668019) according to the manufacturer’s protocol.

Techniques: Comparison, Variant Assay, Control, Activation Assay, Activity Assay, Staining, Expressing, Infection, Labeling, Two Tailed Test, Fluorescence, FACS