Journal: bioRxiv

Article Title: Molecular principles of CRISPR-Cas13 mismatch intolerance enable selective silencing of point-mutated oncogenic RNA with single-base precision

doi: 10.1101/2023.09.26.557083

Figure Lengend Snippet: (A) Silencing efficiency of four perfect-match, BRAF-targeting crRNAs at 48h post-knock-in of WT (grey) or V600E (purple BRAF variants, normalised against crNT. (B) Systematic mutagenesis of non-selective crBRAF-1 was used to engineer V600E-selective crRNAs. Sequences for each crRNA are shown below the barplot; the “U” nucleotide (in red) indicates the uracil in the spacer sequence that basepairs with the target V600E but not with the WT BRAF RNA, and the coloured nucleotides show the position of an additional mismatch introduced at various spacer locations through mutagenesis. Barplot shows silencing efficiency of crBRAF-1 and its mutagenesis derivatives against BRAF-WT (grey) vs BRAF-V600E (purple), normalised against crNT at 48h post-transfection. crE 5 -MM 8,15 and crE 5 -MM 11,15 (orange arrows) show the highest selectivity against BRAF V600E. (C) Delta silencing efficiencies between WT (grey) and V600E (purple) variants for the top-performing crRNAs, indicating the degree of SNV-specificity. Schematics depicting the sequence and basepairing configuration of crE5-MM 8,15 and crE5-MM 11,15 with WT vs V600E-mutant BRAF mRNA targets are shown adjacent. (D) Dose-response curves derived from titration of parental crBRAF-1, crE 5 -MM 8,15 and crE5-MM 11,15 against WT (grey) or V600E (purple) BRAF at 48h post-transfection. For all graphs in A-C, individual data points show averaged fluorescence intensity from eight representative fields of view (n=3 independent experiments), and error bars show mean ± SD. (E) Silencing efficiency assessed by WB in HEK293T cells transfected with PspCas13b and full-length WT or SNV constructs. (F) Silencing efficiency assessed by RT-qPCR in cancer cell lines (A375 melanoma and HCT116 colorectal cancer) which endogenously express the SNV-containing oncogene (mean ± SD from n=3 independent experiments). Statistical significance was determined using unpaired t-tests, where * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Cloning into MSCV-IRES reporter backbones encoding GFP (for BRAF-WT) or mCherry (for BRAF V600E and KRAS) was achieved via plasmid digestion, gel purification, and T4 ligation as previously described • Plasmids encoding full-length BRAF wildtype (p3xFLAG-CMV-BRAF, Addgene #131710) and V600E mutant (p3xFLAG-CMV-BRAF-V600E, Addgene #131723) were gifts from Jacques De Greve. (29)

Techniques: Knock-In, Mutagenesis, Sequencing, Transfection, Derivative Assay, Titration, Fluorescence, Construct, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: TFII-I-mediated polymerase pausing antagonizes GLI2 induction by TGFβ

doi: 10.1093/nar/gkaa476

Figure Lengend Snippet: Knockdown of TFII-I releases RNAPII to transcribe GLI2. ( A ) Real time PCR showing GLI2 mRNA expression in PANC1 cells transfected with NT siRNA or siRNA targeting construct TFII-I and treated with vehicle or flavopiridol, an inhibitor of pause release. ( B ) Immunoprecipitation showing binding of endogenous TFII-I to SPT5. HepG2 cells transfected to express SPT5-HA and TFII-I-FLAG were immunoprecipitated with TFII-I antibody or nonspecific IgG control and then assayed by western blot. ( C ) ChIP assay was performed in HepG2 cells using an anti-NELF-A antibody. The data shows NELF-A I bind the INR of the GLI2 promoter, and that upon TFII-I knockdown endogenous NELF-A binding decreases at this region. These findings indicate TFII-I represses GLI2 gene transcription through RNAPII pausing. For this figure, N = 3 and the asterisk represents P ≤ 0.05.

Article Snippet: Plasmids utilized for experiments included a 3xFLAG-TFII-I expression vector corresponding to the TFII-I isoforms α, β, δ and γ (GenScript, Piscataway, NJ) and SPT5-HA in the p3xFLAG-CMV14 vector (original SPT5 sequence from Capital Biosciences in pORF).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Transfection, Construct, Immunoprecipitation, Binding Assay, Western Blot