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Fig. S10

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ZDB-IMAGE-150915-52
Source
Figures for Gao et al., 2015
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Figure Caption

Fig. S10 Biallelic disruption of topbp1 by CRISP/Cas9 mimics HSPCs deficiency in topbp1cas003 mutant.

(A) Diagram showing the target site of topbp1 gRNA used in this study. The topbp1 gene contains 27 exons. Five gRNAs targeting exon 6, 7 and 9 of topbp1 were designed. The indicated gRNA targeting exon 9 with high mutagenesis rates was used for the following assay. Arrow indicates the target site of the gRNA. Red asterisk represents the position of nonsense mutation in topbp1cas003 mutant. (B) T7 endonuclease I (T7EI) assay showing the mutagenesis efficacy in topbp1 gRNA targeted embryos. topbp1 gRNA (25pg) and nls-zCas9-nls mRNA (150pg) were injected into the wild-type embryos. M, maker; Emb, embryo. (C-D′) The c-myb WISH results showing some of the Cas9 injected wild-type embryos manifested dramatically decreased c-myb expression as same as topbp1cas003 mutant at 4dpf (4/53). (E-F′) The c-myb WISH results showing the Cas9 injected WT/Het embryos displayed dramatically decreased c-myb expression at 4dpf (14/55). Het, topbp1cas003 heterozygote. WT/Het embryos were generated from outcross of topbp1cas003 heterozygote and wild-type fish (Efficiency of CRISPR/Cas9-mediated mutagenesis varies in different microinjection assay). (G-H) Genomic sequencing of the topbp1 gRNA targeting region in the WT or WT/Het embryos (G) and Cas9 injected embryos in D or F (H). Red boxes represent the protospacer-adjacent motif (PAM) site; red arrows indicate the orientation and target site of topbp1 gRNA; blue arrows show the orientation of sequencing. (I) Mutations in 7 out of 9 sequenced topbp1 alleles from a topbp1-targeted F0 embryo. The wild-type reference sequence is underlined. The target site is showed in blue; PAM is highlighted by yellow background. Deletions and insertions are indicated by dashes and lowercase red letters, respectively. The indel mutations are noted at the right of each sequence (+, insertion; , deletion).

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