Journal: bioRxiv

Article Title: Wnt-inducible Lrp6-APEX2 Interacting Proteins Identify ESCRT Machinery and Trk-Fused Gene as Components of the Wnt Signaling Pathway

doi: 10.1101/2020.05.03.072579

Figure Lengend Snippet: CRISPR-Cas9 mediated TFG knock-out inhibits Wnt-dependent β-catenin stabilization and Wnt-induced reporter activity. ( A ) Schematic diagram of Cas9 cleaving the TFG genomic DNA target sequence. The target sequence (which is on the reverse strand) is shown in 5’ to 3’ orientation (left to right); the PAM (protospacer adjacent motif) sequence is in bold. ( B ) Western blot of HEK293T cells confirming complete elimination of TFG protein in Cas9 knock-out cells. β-actin was used as loading control. ( C-F ) Immunostaining for β-catenin on WT or TFG KO cells. Cells were treated with control conditioned medium or with Wnt3a conditioned medium for 3 hours before immunostaining. Note that TFG knock-out decreases Wnt3a-induced β-catenin accumulation; DAPI was used for nuclear counter-staining. Scale bars represent 20 µm. ( G ) Cas9-mediated TFG knock-out reduces response to Wnt3a by 50% in HEK293T BAR/Renilla β-catenin reporter cells. Cells were treated with Wnt3a or control medium for 16 hours before luciferase analysis. ( H ) Luciferase assay of HEK293T BAR reporter cells treated with the GSK3 inhibitor CHIR99021 at 5 µM concentration for 16 hours before being processed for luciferase assay. Note that TFG KO causes an 80% reduction of β-catenin luciferase activity in response to the GSK3 inhibition. Error bars represent standard deviation from triplicate experiments. Statistical significance was calculated with a paired 2-tailed t-Student test. * = P < 0.05; ** = P < 0.01.

Article Snippet: A plasmid based on the original PX459 from Zhang lab, containing Streptococcus pyogenes Cas9 (spCas9)-2A-blasticidin resistance was obtained from Addgene (#118055, from Ken-Ichi Takemaru lab).

Techniques: CRISPR, Knock-Out, Activity Assay, Sequencing, Western Blot, Control, Immunostaining, Staining, Luciferase, Concentration Assay, Inhibition, Standard Deviation

Journal: bioRxiv

Article Title: Wnt-inducible Lrp6-APEX2 Interacting Proteins Identify ESCRT Machinery and Trk-Fused Gene as Components of the Wnt Signaling Pathway

doi: 10.1101/2020.05.03.072579

Figure Lengend Snippet: Genomic DNA was extracted from untransfected WT HEK293T cells, and from a cell clone stably transfected with a Cas9 expression vector containing an sgRNA-encoding sequence specific for human TFG. A 600 nucleotide region spanning the target sequence was amplified by PCR, cloned into sequencing vectors, and analyzed by Sanger sequencing. The reference chromatogram from sequencing results is shown below the DNA sequence. ( A ) Nucleotide sequence of TFG from wild-type (WT) HEK293 cells; the coding frame is indicated by black dots between each codon. The boxed area marks the target sequence recognized by the spacer region in the sgRNA in the WT TFG sequence. The predicted amino acid sequence is shown above the DNA sequence. ( B and C ) Sequences from a TFG knock-out clonal cell line showing the two types of mutant sequences resulting after PCR amplification of mutated genomic DNA. Each TFG allele harbored independent insertion-deletion (indel) events induced by Cas9 as a result of non-homologous end joining (NHEJ) repair. In Mutation 1 (Mut1) Cas9 induced a deletion of a short nucleotide sequence (8 nucleotides, AAGACCCC). Mutation 2 (Mut 2) resulted in the insertion of an extra adenine between the original CCA and AGA codons. Both indel events induced a frameshift mutation, introducing an early STOP codon indicated by an asterisk, generating truncated TFG mutant proteins of approximately 90 amino acids.

Article Snippet: A plasmid based on the original PX459 from Zhang lab, containing Streptococcus pyogenes Cas9 (spCas9)-2A-blasticidin resistance was obtained from Addgene (#118055, from Ken-Ichi Takemaru lab).

Techniques: Stable Transfection, Transfection, Expressing, Plasmid Preparation, Sequencing, Amplification, Clone Assay, Knock-Out, Mutagenesis, Non-Homologous End Joining