Genome editing with use of transcription activator-like effector nucleases (TALENs) to generate null alleles of the zebrafish asxl1 gene. (A) Site-specific targeting for TALEN-directed Fok1 cleavage within exon 2. (B) Nucleotide sequence alignment of the asxl1Δ10 and asxl1Δ11 zebrafish lines compared to wild-type asxl1. Left, TALEN binding site appears in red; right, TALEN binding site appears in green. Dashes in the DNA sequence represent the nucleotides that are deleted during repair of Fok1-induced DNA double-stranded breaks. (C) Targeted Fok1-induced mutagenic lesions in asxl1 produce frameshift mutations that lead to truncated protein products following short regions of novel amino acids, which are indicated in purple. Red ‘X’ denotes stop codons. (D) The truncated Asxl1 protein products predicted to be expressed in the asxl1Δ10 and asxl1Δ11 mutant lines lack all highly conserved functional domains. Red asterisks denote stop codons. (E) asxl1−/− zebrafish larvae mutants appear shorter in length and slimmer than asxl1+/+ and asxl1+/− zebrafish larvae at day 7 and day 9 post-fertilization. Scale bars: 200 µm. (F) Kaplan–Meier survival curves for asxl1+/+, asxl1+/− and asxl1−/− mutants during the first 35 weeks of life. Asterisk (*) at the base of first curve indicates that ∼8% of asxl1−/− fish grow to normal size and survive.
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