Page 267 - Haematologica April 2020

Inflammation and TTP
bred with a wt zebrafish on the double transgenic back- ground to generate F1, F2, and F3 progenies. Sanger sequencing confirmed the presence of various mutations, deletions, and insertions in the region encoding the signal peptide of a13 (Online Supplementary Figure S1B). The CRISPR design tool provided the sgRNA sequence that specifically targets the a13 gene. Any additional off-target was nearly eliminated after multiple generations of out- breeding with the wt zebrafish (Online Supplementary Figure S1C).
The resulting heterozygous siblings with an 8-bp dele- tion in a13 (Figure 1B) were selected for generating a13-/-
zebrafish for our subsequent studies. To facilitate rapid genotyping, we developed a PCR-melting curve strategy. The difference in the melting temperature of amplicons for the area of interest was dramatic between zebrafish with wt and those with a mutated gene (Online Supplementary Figure S2). This strategy provided rapid but reliable genotyping. The 8-nt deletion was predicted to introduce a premature stop within the signal peptide of a13, resulting in no expression of a13 protein, as demon- strated by an automated western blotting system (WES) in the embryo lysates (5 days post-fertilization) and plas- ma of all adult zebrafish tested (Figure 1C). Additionally,
Figure 1. Generation and characterization of a13-/- zebrafish using CRISPR/Cas9. (A) An oligonucleotide con- sisting of a T7 promoter, a gene-specific target sequence (targeting the signal peptide coding region of zebrafish a13), and a guide RNA (gRNA) scaffold sequence was annealed and extended with the gRNA core sequence and then transcribed into a gRNA. (B) Sanger sequencing iden- tified the wt (top) and 8-bp deletion (boxed) in a13-/- F2 progeny zebrafish (bottom, arrowhead). (C) Western blot by a capillary-based WES system demonstrates the absence (a13-/-) and the presence (wt) of a13 protein (∼220 kDa) in the lysate of zebrafish embryos (5 days post-fertilization) and in the plasma of 3-month old zebrafish, respectively. (D) A FRETS-VWF73 assay showed normal cleaving activity in the wt adult zebrafish plasma but not the a13-/-. Such proteolytic activity was abrogated by the addition of 10 mmol/L EDTA. Pooled normal human plasma (NHP) was used as a positive control. Data represent the means ± standard error of mean from three independent experi- ments.
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