(a) Guide RNAs targeting ptch1 or the zebrafish locus syntenic to the human aavs1 safe-harbor locus were co-injected with Cas9 mRNA into zebrafish embryos at the 1-cell stage, genomic DNA was harvested 48h later, and cutting efficiency was assessed by next-generation sequencing. Results are shown for the most efficient gRNAs targeting each locus, which were used in the subsequent generation of transgenic zebrafish. Top line indicates the reference sequence, and individual reads harboring insertion or deletion (In/Del) mutations are shown in decreasing order of abundance. The total fraction of reads with In/Del mutations is shown in parentheses. (b) Schema of experimental design. (c) Kaplan-Meier analysis of zebrafish injected with the CRISPR/Cas9 construct targeting ptch1 or aavs1 (safe harbor control), together with rag2-notch1a^ICD (aavs1, n=27; ptch1, n=26). A two-sided Log-rank (Mantel-Cox) test was used to assess differences in survival between these two groups. (d) Representative ptch1 (top panel) or aavs1 (control) (bottom panel);rag2- notch1a^ICD double transgenic zebrafish at 20 weeks of age. (e) H&E staining of sagittal cross-section (left) and muscle (right) from ptch1 (top) and wild-type (bottom) age-matched zebrafish. (f) Next-generation sequencing of the locus targeted by ptch1 gRNA in a ptch1-mutant rag2- notch1a^ICD double transgenic zebrafish. Note that ptch1 is transcribed from the strand complementary to the gRNA target sequence shown here.

(a) Schema of experimental design. (b) A representative adult ptch1-mutant recipient fish shown at day 7 (top) and 14 (bottom) following transplantation with ptch1; rag2-notch1a^ICD T-ALL. All 32 transplanted ptch1-mutant zebrafish demonstrated similar leukemic engraftment and rapid disease progression. Following leukemic engraftment, fish were treated with 1μM cyclopamine or vehicle control (ethanol) for 14 days. (c) Representative zebrafish in cyclopamine-treated and vehicle control (ethanol) for ptch1-mutant (top panel) and aavs1-control zebrafish (bottom panel) at the indicated time points. (d) Leukemic burden was assessed by mean fluorescence intensity (MFI), with MFI at day 14 normalized to day 0 for each individual fish. Of note, 5 ptch1-mutant fish from each group (vehicle and cyclopamine) met criteria for euthanasia due to radiation toxicity by day 14, and most others became ill soon thereafter, thus the study was terminated at this time point. A two-sided Welch t-test was used to assess differences among treatment groups. (Number of fish per group: ptch1-mutant recipient fish - Day 0, vehicle=10, cyclopamine=11; Day 14, vehicle=5, cyclopamine=5; aavs1-control recipient fish - Day 0, vehicle=9, cyclopamine=11; Day 14, vehicle=8, cyclopamine=9). (e) Cells from a primary T-ALL patient sample that harbored a p.T1106M PTCH1 mutation, which had been engrafted and expanded in immunodeficient mice, were treated with DMSO control, 10nM vismodegib, or 100nM SAG. After 48 hours, (top panel) relative GLI1 mRNA expression (to β-Actin control) and (bottom panel) viability using trypan blue staining were assessed. Results were normalized to DMSO control. Differences were assessed using a Welch t-test.