Figure 7—figure supplement 1.

kdr downstream of TP53^P153Δ predisposes to head embryonal rhabdomyosarcoma (ERMS) in tp53-/- zebrafish.

(A) Schematic of the strategy to target exons 12 and 13 in kdr that contains the enzymatic catalytic domain. (B) Sequence of the targeting guide RNAs targeting exon 12/13 in zebrafish kdr. (C) Polyacrylamide gel showing wild type, mutant heteroduplex, and complete deletion DNA bands obtained from a 641 PCR amplicon of the region of exons 12 and 13 of kdr that was targeted for ablation by three guide RNAs from five independent embryonal rhabdomyosarcoma (ERMS) tumors (T1–T5) from the tp53^-/- + TP53^P153Δ + Cas9/gRNA targeting kdr experimental group. The presence of deletions as visualized by small PCR amplicons corresponding to a loss of 334 bp in a 641 bp wild-type PCR product (deletion mutant amplicon = 212 bp). In addition, presence of heteroduplex products can be seen via PAGE, indicating the presence of indels. (D) Trace DNA sequencing reads from four independent tumors showing that the gRNA used are active and can introduce deletions in exons 12 and 13 of kdr. Indels were confirmed by cloning PCR products and sequencing individual clones. Please see indels +21 bp, –7 bp at the g1 site, 7 bp deletion at the g2 site, and 25 bp deletion at the g3 site. (E) Schematic of the strategy to target exon 2 in mitfa. (F) Sequence of the targeting guide RNAs targeting exon 2 in zebrafish mitfa.