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: bioRxiv

Article Title: Development of potent dual BET/HDAC inhibitors via pharmacophore merging and structure-guided optimization

doi: 10.1101/2023.07.18.547334

Figure Lengend Snippet: Biological effects of the optimized dual BET/HDAC inhibitors. (A) Effect on histone H3 K9/K14 acetylation in Patu8988T cells 48 h after incubation with 1 μΜ compound monitored by Western blot. (B) Western blot showing the concentration-dependent inhibition of histone H3 K9/K14 deacetylation in Patu8988T cells 48 h after treatment with NB503 and NB512. (C) Upregulation of mRNA levels of BET-inhibition biomarkers HEXIM1 and p57 in Patu8988T cells 6 h after treatment with 1 μΜ compound. (D) mRNA levels of oncogenic drivers MYC and TP63 in NMC cells 6 h after treatment with 1 μΜ compound, showing that the optimized dual inhibitors significantly downregulated both transcription factors. (E) Cell viability of pancreatic cancer cell line PatuT ( left ) and NMC cell line HCC2429 ( right ) after 3d-treatment with different concentrations of dual BET/HDAC inhibitors.

Article Snippet: Nut midline carcinoma cell line HCC2429 was kindly provided by Lead Discovery Center GmbH (Dortmund, Germany) and was cultured in RPMI1640 medium containing 10% FBS, 2 mM L-glutamine and 1% penicillin-streptomycin.

Techniques: Incubation, Western Blot, Concentration Assay, Inhibition