FIGURE

Figure 1—figure supplement 1—source data 1.

ID

ZDB-FIG-220507-23

Publication

Thumberger et al., 2022 - Boosting targeted genome editing using the hei-tag

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Figure 1—figure supplement 1—source data 1.

Raw data for quantifications shown in <xref rid='fig1s1' ref-type='fig'>Figure 1—figure supplement 1</xref>.
Identification of the hei-tag. Comparison of OlOca2 knock-out efficiency (quantification of eye pigmentation) using a permutation screen of peptide domains (nuclear localization signals [NLSs], Myc-tag, amino acid linkers) flanking a mammalian codon-optimized Cas9 enzyme. Injection mix contained 30ng/µl OlOca2 T1, T2, 150ng/µl tagged Cas9 variant mRNA, 20ng/µl GFP mRNA injection marker. Particular peptides and relative positions indicated by schematics. Constellation of peptides sorted by knock-out efficiency. The ‘hei-tag’ myc-flexible-linker-oNLS-Cas9-oNLS (heiCas9) variant was identified being most efficient. JDS246-Cas9 (Addgene #43861), MSI-Cas9-Xl (myc-Cas9) was cloned following Zhang et al., 2014. Peptides used: FLAG, FLAG tag; F, flexible linker; I, internal linker; M, cMyc-tag; O, optimized NLS (Inoue et al., 2016); S, SV40 NLS (Kalderon et al., 1984); Xl, bipartite Xenopus laevis nucleoplasmin NLS (Dingwall et al., 1988). For sequences, see Supplementary files 1 and 2. 0 = fully pigmented, 255 = completely unpigmented; n, number of eyes analyzed. Figure 1—figure supplement 1—source data 1.

Expression Data

Expression Detail

Antibody Labeling

Phenotype Data

Phenotype Detail

Acknowledgments

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