Generation of rnaseh2a−/− knockout zebrafish and initial characterization. (A) Protein alignment between zebrafish, Danio rerio (Dr) and Homo sapiens (Hs) RNaseH2a. In the zebrafish protein, the amino acids that are lost as a result of the mutation are greyed out, the last homologous amino acid is highlighted red. Asterisk show sites of human AGS mutations, a critical tyrosine required highlighted in red. (B) WISH staining shows universal RNaseH2a expression in wild-type zebrafish embryos at 24hpf, lower image shows sense probe as control. (C) In exon 6 (red), a 49bp deletion was created utilizing the CRISPR/Cas9 system. Exon 6 sequence is shown below with deleted bases in red. The deletion results in a premature stop codon and truncated protein of 205aa in length. (D) Embryos resulting from an rnaseh2a+/− incross do not show any visible phenotype and are able to develop to adulthood with no external phenotype. NB. spotty pigment is due to a background mutation in laboratory zebrafish. (E) Inheritance of the rnaseh2a mutation is in a homozygous recessive manner at 24hpf and displays mendelian inheritance. X2 = 2.66, with two degrees of freedom; two-tailed P value of 0.529.
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