Figure 4
- ID
- ZDB-FIG-220507-27
- Publication
- Thumberger et al., 2022 - Boosting targeted genome editing using the hei-tag
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Phenotypic range and quantification of heiBE4-Gam-mediated C-to-T transitions in medaka embryos. (a) Categories of typically observed loss-of-pigmentation phenotypes in oca2 editants. The observed pigmentation phenotypes range from (almost) unpigmented eyes, that is, a very strong knock-out (top panel) over intermediate (central panel) to no loss of pigmentation (bottom panel). Quantification of phenotype resulting from injections with either BE4-Gam or heiBE4-Gam (red) mRNA and OlOca2 T1, T3, or T4 sgRNAs. Note: dramatic increase of bi-allelic knock-out rate when using heiBE4-Gam. n, number of eyes analyzed. Control median = 0.0; medians BE4-Gam vs. heiBE4-Gam: OlOca2 T1, 0.6 vs. 28.0, p = 1.737; OlOca2 T3, 0.0 vs. 0.8, p = 0.0471; OlOca2 T4, 93.8 vs. 170.1, p = 5.215e-12. Bold lines, median values. Statistical analysis performed in R, pairwise Wilcoxon rank sum test. (b) Schematic representation of base editing window in OlOca2 T1 target site (PAM, protospacer adjacent motif). C-to-T transition of C995 and C996 edits the threonine (T) codon to isoleucine (I) (T332I); C997T creates a pre-mature STOP codon (Q333*). Nucleotide positions refer to the oca2 open reading frame. (c) Quantification of Sanger sequencing reads at nucleotides C995, C996, C997 inside the base editing window of three injected embryo pools (five embryos each) reveals overall dramatic increase of C-to-T base transition when using heiBE4-Gam. Note 1.7-fold increase of C997T transition, that is, efficient introduction of a pre-mature STOP codon. Mean values indicated by bold horizontal lines, Figure 4—figure supplement 1.
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