Fig. 2

(A) Sequence of oligonucleotide used for guide RNA (gRNA) synthesis. The scaffolding sequence is in purple; the target sequence is in green, and the T7 polymerase promoter is in blue. (B) Intron-exon structure of the D. rerio tdp1 gene. Exon 2 (in red) was targeted for mutation by Cas9; scale bar, 5000 bp. Photo credit: Ringaile Zaksauskaite, University of Sheffield (generated using http://wormweb.org/exonintron). (C) Sequences of the target region in tdp1^WT zebrafish and two isolated deletion alleles, tdp1^SH475 and tdp1^SH476. The 5-bp deletion (SH476; light blue box) and the 4-bp deletion (SH475; green square). (D) Tdp1^−/+ zebrafish were crossed and genotyped at adulthood; χ² = 2.941 with two degrees of freedom; two-tailed P value of 0.5316. (E) Diagram depicting the TDP1 activity assay showing a 5′ labeled oligomer with a 3′-phosphotyrosyl (PY) that is incubated with zebrafish protein lysate. Active TDP1 processes the 3′-PY into a phosphate group, resulting in a band shift on a DNA sequencing gel. (F) TDP1 activity assay was performed on 600 ng of lysate from 4-dpf embryos. (G) TDP1 activity assay was performed on fin clips from adult zebrafish. LB, lysis buffer control