Targeted FGFR4 Manipulation via CRISPR-Based Tools

For CRISPRi repression of FGFR4, we constructed sgRNAs targeting the FGFR4 promoter sequence and dCas9-KRAB fusion protein using a lentivirus-based plasmid^{75 (link)} (Addgene, #71236). For programmable m6A modification of intracellular FGFR4 mRNA with a Cas13-directed methyltransferase^{76 (link)}, we constructed sgRNAs targeting the upstream FGFR4 mRNA m6A sites using the pC0043-PspCas13b crRNA backbone (Addgene, #103854). Fusion of dCas13, METTL3, and METTL14 proteins was used as an m6A methyltransferase tool (Addgene, #155367). The sequences of the sgRNAs are listed in Supplementary Table 7. The SLC7A11/FPN1 promoter region (−1 to −2000 from the transcriptional start site) was amplified using hot-start DNA polymerase (Takara, RR006A). To construct a promoter-luciferase reporter, we cloned the promoter fragment into the pGL3-basic vector. Site-specific mutation of the SLC7A11/FPN1 promoter region luciferase reporter construct was generated using a Site-Directed Mutagenesis Kit (YEASEN). The sequences of all the vectors were validated by Sanger sequencing. Cells were transfected using Lipofectamine 3000 (Invitrogen).