Base editor in zebrafish using a whole zebrafish codon-optimized BE4 system. a Schematic diagram of the zAncBE4max system. The system consists of whole zebrafish codon-optimized genes/components, including rat deaminase APOBEC1(red), Cas9n D10A nickase (green), two uracil DNA glycosylase inhibitors (purple), and a bipartite NLS at both ends, which convert cytidine to thymine within a window of − 17 to − 13 bases from the PAM of the gRNA. Both zAncBE4max and gRNA are linked to the T7 promoter for in vitro transcription. Prefix “z” indicates zebrafish codon-optimized components. b The targeted genes and corresponding sites designed and used in this study. PAM region (blue); target cytidines (red)

Base C-to-T editing efficiency using zAncBE4max in zebrafish. The zAncBE4max system (right panel) showed higher activity of C-to-T editing than the BE3 system (left panel) at 6 tested sites. Red letter C indicates sites of C-to-T conversion and corresponding overlapped peaks in sequencing chromatogram. PAM region is indicated in blue. Mutant number in sequenced clones is shown on the right of sequences

The on-target C-to-T editing efficiency of BE3 and zAncBE4max at 6 target sites. a Percentage of each C-to-T conversion along the target sequence for comparison of BE3 and zAncBE4max. b Overall C-to-T editing efficiency for BE3 and zAncBE4max at each C of the 6 target sites. Mann-Whitney U test was performed. n = 3 biologically independent samples for each target

Generation of an AMS zebrafish model using the zAncBE4max system. a The designed target site in the twist2 gene on chromosome 9 in zebrafish. The nucleotide changes in C-to-T editing are underlined in red. The corresponding amino acid change is E78K. PAM region is indicated in blue. b Homozygous mutant (E78K) embryos exhibit a short truck and curved tail (red arrowheads) at 48 hpf. Scale bar, 100 μm. c Genotyping of homozygous mutant larva by PCR and sequencing. Red arrow indicates point mutation site C-to-T. d Phenotype of heterozygous mutants (E78K) exhibited a protruding jaw, unclosed mouth (red arrows), and emaciated body at the age of 6 months. For more details, see Additional file 3: Movie 1. e qRT-PCR analysis indicates the relative expression levels of twist2 and related genes in twist2 homozygous E78K mutant embryos

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