p3xflag cmv14 braf wt  (Addgene inc)

Journal: Science Advances

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

doi: 10.1126/sciadv.adl0731

Figure Lengend Snippet: ( A ) Silencing efficiency of four perfect-match, BRAF -targeting crRNAs at 48 hours after knock-in of WT (gray) or V600E (purple BRAF variants, normalized against crNT. ( B ) Systematic mutagenesis of nonselective cr BRAF -1 was used to engineer V600E -selective crRNAs. Sequences for each crRNA are shown below the bar plot; the “U” nucleotide (in red) indicates the uracil in the spacer sequence that base pairs with the target V600E but not with the WT BRAF RNA, and the colored nucleotides show the position of an additional mismatch introduced at various spacer locations through mutagenesis. Bar plot shows the silencing efficiency of cr BRAF -1 and its mutagenesis derivatives against BRAF - WT (gray) versus BRAF - V600E (purple), normalized against crNT at 48 hours after 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 (gray) and V600E (purple) variants for the top-performing crRNAs, indicating the degree of SNV-specificity. Schematics depicting the sequence and base-pairing configuration of crE 5 -MM 8,15 and crE 5 -MM 11,15 with WT versus V600E -mutant BRAF mRNA targets are shown adjacent. ( D ) Dose-response curves derived from titration of parental cr BRAF -1, crE 5 -MM 8,15 , and crE 5 -MM 11,15 against WT (gray) or V600E (purple) BRAF at 48 hours after transfection. For all graphs in (A) to (C), individual data points show averaged fluorescence intensity from eight representative fields of view ( n = 3 independent experiments), and error bars show means ± SD. ( E ) Silencing efficiency assessed by Western blotting in HEK293T cells transfected with Psp Cas13b 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 (means ± SD from n = 3 independent experiments). Statistical significance was determined using unpaired t tests, where ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: Plasmids encoding full-length BRAF WT (p3xFLAG-CMV- BRAF , Addgene #131710) and V600E mutant (p3xFLAG-CMV- BRAF - V600E , Addgene #131723) were gifts from J.

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

Journal: Science advances

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

doi: 10.1126/sciadv.adl0731

Figure Lengend Snippet: Fig. 7. SNV-selective silencing of BRAF V600E using orthogonal PspCas13b. (A) Silencing efficiency of four perfect-match, BRAF-targeting crRNAs at 48 hours after knock-in of WT (gray) or V600E (purple BRAF variants, normalized against crNT. (B) Systematic mutagenesis of nonselective crBRAF-1 was used to engineer V600E-selective crRNAs. Sequences for each crRNA are shown below the bar plot; the “U” nucleotide (in red) indicates the uracil in the spacer sequence that base pairs with the target V600E but not with the WT BRAF RNA, and the colored nucleotides show the position of an additional mismatch introduced at various spacer locations through mutagen- esis. Bar plot shows the silencing efficiency of crBRAF-1 and its mutagenesis derivatives against BRAF-WT (gray) versus BRAF-V600E (purple), normalized against crNT at 48 hours after transfection. crE5-MM8,15 and crE5-MM11,15 (orange arrows) show the highest selectivity against BRAF V600E. (C) Delta silencing efficiencies between WT (gray) and V600E (purple) variants for the top-performing crRNAs, indicating the degree of SNV-specificity. Schematics depicting the sequence and base-pairing configu- ration of crE5-MM8,15 and crE5-MM11,15 with WT versus V600E-mutant BRAF mRNA targets are shown adjacent. (D) Dose-response curves derived from titration of parental crBRAF-1, crE5-MM8,15, and crE5-MM11,15 against WT (gray) or V600E (purple) BRAF at 48 hours after transfection. For all graphs in (A) to (C), individual data points show averaged fluorescence intensity from eight representative fields of view (n = 3 independent experiments), and error bars show means ± SD. (E) Silencing efficiency as- sessed by Western blotting 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 (means ± SD from n = 3 independent experiments). Statistical significance was determined using unpaired t tests, where **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Article Snippet: Plasmids encoding fulllength BRAF WT (p3xFLAG- CMV- BRAF, Addgene #131710) and V600E mutant (p3xFLAG- CMV- BRAF- V600E, Addgene #131723) were gifts from J.

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