Prime editing substitution in the zebrafish crbn gene, comparing Cas9-nickase-based and nuclease-based Prime Editors.

a, Schematic illustration of the functioning of prime editing by the Cas9-nickase-based Prime Editor (PE2) and the nuclease-based Prime Editor (PEn). b-d, Comparison between PEn and PE2 in prime editing substitution in the crbn gene with the pegRNA refolding procedure. Proportions of editing outcomes in individual injected embryos are shown by amplicon sequencing (b), alongside quantitative analyses of precise prime edits (c) and indels (d) comparing experimental conditions (n = 10 per group). P-values were determined using one-way ANOVA with Tukey’s multiple comparison test in (c), and the Kruskal-Wallis test with Dunn’s multiple comparison test in (d). Error bars in the bar graphs represent the mean and standard deviation, and individual data points indicate values from single injected embryos.

Prime editing insertion in zebrafish ror2 gene using Cas9-nuclease-based Prime Editor.

a, Schematic illustration of the functional domains in the Ror2 protein and alignment of partial amino acid sequences within the tyrosine kinase domain. Sequences from multiple species, including those related to Robinow syndrome (RS) in humans W720X and the corresponding zebrafish W722X mutant, are aligned. The conserved tyrosine residue is highlighted. b, Schematic illustration of guide RNA (gRNA) designs for prime editing insertion in ror2. c, Schematic illustration of prime editing insertion by Cas9-nuclease-based Prime Editor (PEn). An additional DNA fragment, reverse-transcribed at the target cleavage site, containing the programmed insertion, is integrated into the genome via homology-directed repair or non-homologous end joining. d, Agarose gel images of genomic PCR products from embryos injected with Prime Editor mRNA and pegRNA/springRNA. PCR products of the ror2 target region (top) and those after digestion with T7 endonuclease I (T7E1, bottom). e, Sequence alignment of the edits in the ror2 target site obtained from embryos injected with PEn/springRNA. Prime editing insertion (TGA) is outlined, and the gRNA target sequence is underlined. f, Quantitative comparison of editing outcomes using different combinations of Prime Editor and gRNA. The proportion of sequence reads with each type of edit in amplicon sequencing is presented.

Comparing different prime editing approaches in zebrafish embryos.

a-c, Comparison between PEn and PE2 in prime editing insertion into ror2 with various delivery methods. The proportion of editing outcomes in individual injected embryos was assessed through amplicon sequencing (a), along with a quantitative analysis of precise prime edit (b) and indels (c) across experimental conditions (n = 10 per group). d, Sequence alignment of edits in prime editing insertion in ror2 via the PEn mRNA/springRNA combination. e, Schematic illustration of springRNA design featuring an abasic RNA spacer for prime editing insertion in ror2. f and g, Proportion of reads with scaffold incorporation (f) and precise prime edit (g) as determined by amplicon sequencing of prime editing insertion in ror2 using control and abasic springRNA (n = 10 per group; one sample of the PEn RNP/abasic springRNA combination was excluded from analysis due to low read count). h and i, Prime editing insertion in the adgrf3b gene using PEn. The proportion of reads with a precise 3 bp (h) and 12 bp insertion (i) was evaluated using pegRNA or springRNA (n = 10 per group). P-values were determined by Welch’s one-way ANOVA with Dunnett T3 multiple comparison test in b and c, and Kruskal-Wallis test with Dunn’s multiple comparison test in f and g. Error bars in bar graphs represent the mean and standard deviation, and each individual data point indicates the value from a single injected embryo.

Generation and characterisation of stable ror2W722X mutant.

a, Summary of the screening of F1 embryos. Sixteen embryos were genotyped per F0 founder. Each circle represents the genotype of a single embryo. The embryos were obtained by outcrossing the injected founder with wild-type fish; thus, all embryos with the mutation are heterozygous. b-d, Lateral images of wild type (b), zygotic ror2W722X mutant (c), and maternal-zygotic (MZ) ror2W722Xmutant (d) larvae at 5 days post-fertilisation (dpf). e, Sanger sequencing chromatogram of wild type (top) and ror2W722X mutant (bottom) at the prime editing target site in ror2. Prime editing insertion (TGA) is highlighted. The target sequence of the guide RNA is underlined, and the cleavage site of Cas9 is indicated by a dotted line. f, Quantitative analysis of total length comparing wild type, zygotic ror2W722X mutant, and MZ ror2W722X mutant at 5 dpf. Each data point on the graph represents the value from a single larva (n = 10 per group). P-values were determined by one-way ANOVA with a Tukey multiple comparisons test. Error bars represent the mean and standard deviation.

Prime editing to insert a nuclear localisation signal sequence into the smyhc1:gfp transgene.

a, Schematic representation of the prime editing insertion of the nuclear localisation signal (NLS) sequence into the smyhc1:gfp transgene and the expected eGFP expression in slow-twitch muscle fibres. b, Confocal microscopy image of the trunk muscle in smyhc1:gfp larvae at 4 days post-fertilisation (dpf) that were injected with the PEn RNP complex for the prime editing NLS insertion. Putative nuclear GFP expression is indicated by an arrowhead. Anterior is positioned to the left. c, Quantitative analysis of the efficiency of precise NLS insertion via amplicon sequencing. Two pegRNAs of differing lengths are compared (n = 10 per group, with one sample excluded from the analysis due to low read count). The p-value was determined using the Mann-Whitney U test. Error bars represent the mean and standard deviation, while individual data points on the graph indicate values from single injected larvae. d, Confocal microscopy images of the trunk muscle of F1 larvae exhibiting nuclear GFP expression at 2 dpf, obtained from founder 1 (see panel f). The GFP fluorescence channel (left) and pseudo-colour (right) images are shown. Anterior is to the left. e, Sequence alignment of the edits in the target site obtained from F1 embryos exhibiting nuclear GFP expression (founder 1). The NLS sequence is outlined in red. f, Summary of F1 embryo screening. F1 embryos obtained from 6 founders are categorised based on the GFP expression pattern. The embryos were produced by outcrossing the heterozygous smyhc1:gfp founder injected with the PEn RNP complex to wild type; thus, half of the embryos are expected to be negative for the smyhc1:gfp transgene.