Fig. S10

Fig. S10 Biallelic disruption of topbp1 by CRISP/Cas9 mimics HSPCs deficiency in topbp1^cas003 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 topbp1^cas003 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 topbp1^cas003 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, topbp1^cas003 heterozygote. WT/Het embryos were generated from outcross of topbp1^cas003 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).