Fig. 3

Precise cytosine base editing by zevoCDA1-NL and zevoCDA1-198.a The mRNA construct of zevoCDA1-NL and zevoCDA1-198 for precise cytosine base editing. The XTEN linker was deleted from zevoCDA1-SpRY-BE4max to generate zevoCDA1-NL, and the nuclear export signal (NES) located at the C-terminus of the evoCDA1 deaminase was subsequently removed to generate zevoCDA1-198. bpNLS: bipartite nuclear localization, zevoCDA1: cytosine deaminase, nSpRYCas9: SpCas9 nickase varient, linker: SGGSSGGS amino acid, UGI: Uracil glycosylase inhibitor. b?d Assessment of the editing efficiency and targeting window of zevoCDA1-SpRY-BE4max (b), zevoCDA1-NL (c) and zevoCDA1-198 (d) using 8 gRNAs targeting NNN PAMs. The data represent the sum of three independently replicated experiments, and the error bars represent the standard deviation of the mean values. e Summary of editing efficiency of zevoCDA1-SpRY-BE4max, zevoCDA1-NL and zevoCDA1-198 at 10 NNN PAM sites across 8 genes. The dotted range indicates the editing window. f Schematic diagram of the pitx2 target locus. The target sequence is displayed with the PAM highlighted in red and the target nucleotide and expected nucleotide changes are highlighted in blue. g Comparison of Sanger sequencing results for zevoCDA1-SpRY-BE4max, zevoCDA1-NL, and zevoCDA1-198 at the pitx2 R89W target locus. The red arrowhead indicates the expected nucleotide substitutions. h?i? Dorsal view of 5 dpf F1 homozygous embryos with the pitx2 R89W mutation showing absence of anterior chamber (ac). WT anterior chambers are highlighted with black arrows in the close-up view of the head (h) and with a dashed outline and black arrow in the close-up view of the eye (h?). The absence of the anterior chamber in mutant zebrafish is highlighted by a white arrow in the close-up view of the head (i) and eye (i?). Scale bars: 100 ?m. j?k? Alcian blue staining of 5-dpf wild-type and pitx2 R89W mutant embryos. The ventral view (j, j?) and lateral view of (k, k?) wild-type AB zebrafish (j, k) and pitx2 R89W (j?, k?) embryos at 5 dpf. pitx2 R89W mutant (j?, k?) shows severe structural malformations in Meckel?s cartilage (mc) highlighted with arrow and red dotted line compared with WT AB (j, k). Scale bars: 200 ?m. Three independent experiments were repeated with similar results. Source data are provided as a Source Data file.