FIG. 8.

Modeling mutations in the zebrafish mt-tl1 gene. (A) Protospacer region highlighting the putative cytosine targets in the mt-tl1 locus. Restriction site for the enzyme SacI is marked by a dotted rectangle. (B) Screening of embryos harboring mutations by RFLP. C-to-T editing event in the target locus leads to loss of restriction site for the SacI restriction enzyme. The presence of undigested WT bands of expected size, 186 and 177 bp (marked by red arrow), is observed in control embryos. PCR amplicons from the injected embryos show the undigested band, indicated by the loss of restriction site postediting (marked by white asterisk*). Each lane represents an individual embryo. (C) Editing efficiency for the favorable 5′TC sites present in the protospacer region, both in mutant and control zebrafish embryos. Control embryos and embryos harboring desired C-to-T edits are highlighted by black and red color circles, respectively. Each data point represents an individual zebrafish embryo. Data are represented from independent experiments (Injected: N = 150 embryos and were screened and N = 10 embryos [RFLP positive] were genotyped by Sanger sequencing; Control = 60 embryos were screened and N = 7 embryos were genotyped by Sanger sequencing for which data have been included in the graph). (D) Representative chromatogram of the control and mutant embryos indicating the percentage of editing at each cytosine residue in the protospacer region. Asterisk (*) denotes the site of edit with corresponding editing percentage (C-to-T or G-to-A). Chromatograms and editing table plot were obtained using EditR. (E) Percentage of edited alleles with C-to-T edits at different cytosine residues obtained from deep sequencing. Representative data from embryo 4 show the editing frequency of cytosine (antisense strand) residue (or guanine on the sense strand), which is highlighted in red and circled. The combination of left arm- pT3-FusXTBE-N and right arm- pT3-FusXTBE-C was used to obtain the edits. Color images are available online.