Journal: Cell Genomics

Article Title: Optimization of Cas12a for multiplexed genome-scale transcriptional activation

doi: 10.1016/j.xgen.2023.100387

Figure Lengend Snippet: Nanobody recruitment improves CRISPRa activity (A) Schematic representing dCas12a nanobody-based recruitment of the transactivation domain (top). Plasmid maps depicting one vector containing the ALFA tag fused to dCas12a and a second vector containing the ALFA nanobody, TAD, and targeting guide (bottom). (B) Heatmaps displaying comparative performance between the p65, VP64, and Activ TADs when recruited via nanobody to dCas12a with increasing numbers of ALFA tags (1×, 3×, 5×) at either the N or the C terminus (N′ or C′). Variable vector components are indicated by dashed lines. Experiments conducted in HT29, HCC2429, and A375 cell lines. Color scale reflects levels of normalized MFI of CD4 expression within each cell line. (C) Bar plot illustrating combinatorial effects emerging from the direct tethering of TADs to the N terminus of dCas12a and nanobody-based recruitment of varying TAD configurations to the N and/or C termini in HT29 cells. The x axis shows normalized MFI values of CD4 expression on a log10 scale. (D) Bar plot comparing CRISPRa activity when a nanobody-TAD combination is recruited to the N′ or C′ terminus of Cas12a tethered to VP64 (N′ 5× tag, C′ 5× tag). Normalized CD4 MFI values are shown for three cell lines (A375, HT29, and HCC2429). See also Figure S2 .

Article Snippet: A375 (female), HCC2429 (female), HT29 (female), and MelJuSo (female) cells were obtained from the Cancer Cell Line Encyclopedia at the Broad Institute.

Techniques: Activity Assay, Plasmid Preparation, Expressing

Journal: Cell Genomics

Article Title: Optimization of Cas12a for multiplexed genome-scale transcriptional activation

doi: 10.1016/j.xgen.2023.100387

Figure Lengend Snippet: Effective multiplexing with a nanobody-based system (A) Schematic depicting single-gene targeting guide cassette architecture (top). Histograms show expression levels of CD4 (APC), CD274 (APC), CD97 (FITC), and CD26 (FITC) in HT29, A375, and HCC2429 cells expressing 5×-tag-dCas12a-VP64 when targeted individually by three guides per gene paired with nanobody-VP64, nanobody-p65, or nanobody-p65-HSF1 (bottom). Data from one representative replicate shown; data for all replicates are included in . (B) Schematic depicting multiplexed targeting guide cassette architecture (top). Heatmaps of normalized MFI values for CD26, CD4, CD97, and CD274 in HT29, A375, and HCC2429 cells expressing 5×-tag-dCas12a-VP64 when targeted simultaneously by one guide per gene paired with different TADs, same as those used in (A). MFI values were normalized to basal expression within each cell line/gene combination, as in the color scale (bottom). (C) Comparison of RNA expression levels across samples expressing 5×-tag-dCas12a-VP64 and either nanobody-VP64 or nanobody-p65 with or without three CD4-targeting guides. Shrunken LFC in the CD4-targeting population is plotted against mean normalized read counts of all replicates for baseline expression (n = 3). See also Figure S3 .

Article Snippet: A375 (female), HCC2429 (female), HT29 (female), and MelJuSo (female) cells were obtained from the Cancer Cell Line Encyclopedia at the Broad Institute.

Techniques: Multiplexing, Expressing, Comparison, RNA Expression

Journal: Cell Genomics

Article Title: Optimization of Cas12a for multiplexed genome-scale transcriptional activation

doi: 10.1016/j.xgen.2023.100387

Figure Lengend Snippet:

Article Snippet: A375 (female), HCC2429 (female), HT29 (female), and MelJuSo (female) cells were obtained from the Cancer Cell Line Encyclopedia at the Broad Institute.

Techniques: Recombinant, Biomarker Discovery, Library Amplification, Software

Journal: Oncogene

Article Title: Therapeutic targeting of p300/CBP HAT domain for the treatment of NUT midline carcinoma

doi: 10.1038/s41388-020-1301-9

Figure Lengend Snippet: a Chemical probe screening in three tumor cell lines. HCC2429, NUT midline carcinoma; Patu8988T, pancreatic ductal adenocarcinoma; QGP-1, pancreatic neuroendocrine tumor. Cells were incubated with each of the chemical probes at a concentration of 10 µM for 72 h and cell viabilities were measured by CellTiter Glo Cell Viability assay. The values were normalized to dimethyl sulfoxide (DMSO)-treated samples and a heatmap was generated based on the mean values of three independent experiments. The heat map was colored according to normalized cell viability as depicted in the figure capture. The p -values of positive hits (JQ1, A-485 and BTOZ-1) were presented in the text. b Venn diagram analysis showing NMC-selective and -unselective inhibitors. Probes with cell viability less than 50% in at least one cell line from the screening above were chosen as potent hits. c IC 50 of A-485 on three NMC cell lines and six cell lines of other tumor identities. Mean ± SEM from three independent experiments, * P ≤ 0.05. d Comparison of the growth effects A-485 (red circles) and the inactive analogue A-486 (black square) on three NMC cell lines. IC 50 of A-485 is shown in the graph. Mean ± SD from three technical replicates. In ( c ) and ( d ), cells were incubated with inhibitors at a concentration range between 10 nM and 25 µM. Cell viability was monitored after 72 h by CellTiter Glo Cell Viability assay. The dose response curve was used to determine the IC50 by Prism.

Article Snippet: HCC2429, Ty-82 and 00–143 were kindly provided from Lead Discovery Center GmbH (Dortmund, Germany).

Techniques: Incubation, Concentration Assay, Viability Assay, Generated, Comparison

Journal: Oncogene

Article Title: Therapeutic targeting of p300/CBP HAT domain for the treatment of NUT midline carcinoma

doi: 10.1038/s41388-020-1301-9

Figure Lengend Snippet: a Immunofluoresence detection of H3K27ac, BRD4-NUT and BRD4 wt proteins in HCC2429 cells incubated with 1 µM A-485 or DMSO for 3 days. Scale bar = 10 µm. b Chromatin immunoprecipitation (ChIP) analysis of H3K27ac and BRD4-NUT at the MYC promoter and TP63 enhancer regions in HCC2429 cells incubated with 1 µM A-485 or DMSO for 3 days. Chromatin was precipitated with normal rabbit IgG (IgG as control), H3K27ac and NUT antibodies. Precipitated chromatin was analyzed using qPCR and presented as fold enrichment to IgG control. Mean ± SEM from four independent experiments, ** P ≤ 0.01, * P ≤ 0.05. c Quantitative RT-PCR analysis of MYC , CCAT1 and TP63 genes and ( d ) immunoblot analysis of H3K27ac and MYC proteins in HCC2429 cells incubated with A-485 at indicated concentrations for 48 h. Mean ± SEM from three independent experiments, *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05; n.s., not significant.

Article Snippet: HCC2429, Ty-82 and 00–143 were kindly provided from Lead Discovery Center GmbH (Dortmund, Germany).

Techniques: Incubation, Chromatin Immunoprecipitation, Control, Quantitative RT-PCR, Western Blot

Journal: Oncogene

Article Title: Therapeutic targeting of p300/CBP HAT domain for the treatment of NUT midline carcinoma

doi: 10.1038/s41388-020-1301-9

Figure Lengend Snippet: a Hemacolor staining of HCC2429 cells incubated with 0.5 or 1 µM A-485 for 5 days. b Immunofluoresence detection of cytokeratin in HCC2429 cells incubated with 0.5 µM A-485 or JQ1 for 5 days. Scale bar = 20 µm. c Quantitative RT-PCR analysis of squamous tissue genes ( KRT10 , KRT14 and TGM1 ) and c-fos in HCC2429 cells incubated with 0.5 or 1 µM A-485 or 0.5 µM JQ1 for 5 days. Mean ± SEM from three independent experiments, *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05. d Immunoblot analysis of Involucrin in HCC2429 cells incubated with 0.5 or 1 µM A-485 or 0.5 µM JQ1 for 5 days. e Flow cytometry analysis of HCC2429 cells incubated with 0.5 or 1 µM A-485 for 24, 48 and 72 h. Mean ± SEM from three independent experiments, *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05. f Immunoblot analysis of cleaved caspase-3 in HCC2429 cells incubated with 0.5 or 1 µM A-485 for 24, 48 and 72 h.

Article Snippet: HCC2429, Ty-82 and 00–143 were kindly provided from Lead Discovery Center GmbH (Dortmund, Germany).

Techniques: Staining, Incubation, Quantitative RT-PCR, Western Blot, Flow Cytometry

Journal: Oncogene

Article Title: Therapeutic targeting of p300/CBP HAT domain for the treatment of NUT midline carcinoma

doi: 10.1038/s41388-020-1301-9

Figure Lengend Snippet: a Combination response to A-485 and JQ1 for HCC2429 cells. CellTiterGlo cell viability assay was performed to measure cell viabilities of all the indicated dose combinations for 72 h. Synergy effects were evaluated using SynergyFinder ( https://synergyfinder.fimm.fi ). The ZIP synergy score is averaged over all the dose combination cells. b , c Hierarchical clustering ( b ) and Venn diagram analysis ( c ) of the differentially expressed genes in HCC2429 cells treated with 50 nM JQ1 and 250 nM A-485 alone or combined for 8 h. Each treatment was done in triplicate. d Representative GSEA plots showing significantly enriched up- and downregulated pathways (combination-treatment versus DMSO). e Immunoblot analysis of cleaved caspase-3 in HCC2429 cells incubated with 50 nM JQ1 and 250 nM A-485 alone or combined for 72 h. f Colony formation assay for HCC2429 cells incubated with 50 nM JQ1 and 250 nM A-485 alone or combined for 72 h. g Quantitative RT-PCR analysis of squamous tissue genes ( KRT10 , KRT14 and TGM1 ) and c-fos in HCC2429 cells incubated with 50 nM JQ1 and 250 nM A-485 alone or combined for 5 days. Mean ± SEM from three independent experiments, *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05; n.s., not significant.

Article Snippet: HCC2429, Ty-82 and 00–143 were kindly provided from Lead Discovery Center GmbH (Dortmund, Germany).

Techniques: Viability Assay, Western Blot, Incubation, Colony Assay, Quantitative RT-PCR