Fig. S2
Tissue-specific expression of Cas9 and gene targeting with the CRISPR vector (related to Figure 2). (A) Sequence of the portion of the vector presented in Figure 2A encompassing the U6 promoter and the gRNA scaffold. (B) Representative images of whole mount in situ hybridization (WISH) using an anti-sense RNA probe against Cas9 mRNA in 48 hpf embryos injected with Tol2 mRNA and the pcmlc2:GFP, U6:gRNA vectors expressing Cas9 under the control of the indicated promoters. (C) T7E1 mutagenesis assay at the CRISPR target sites in the p53 or urod genes. The assay was performed on genomic DNA from the blood of 2 dpf embryos injected at the one-cell stage with Tol2 mRNA and the pcmlc2:GFP, U6:gRNA, gata1:Cas9 vectors containing a gRNA against p53 or urod. Arrows point to cleavage bands. (D) Most frequent (>2%) mutant urod and p53 alleles found by deep-sequencing in the blood of embryos injected with Tol2 mRNA and the pcmlc2:GFP, U6:gRNA, gata1:Cas9 vectors containing gRNAs against urod and p53 respectively. The CRISPR target sequence is in green, mutations in red. The type of mutation is indicated (del: large deletion). (E) T7E1 mutagenesis assay at the CRISPR target site in the urod gene. The assay was performed on genomic DNA from sorted mCherry-positive cells from 2 dpf Tg(mylz2:mCherry) embryos injected at the one-cell stage with Tol2 mRNA and the pcmlc2:GFP, U6:gRNA urod, mylz2:Cas9 or pcmlc2:GFP, U6:gRNA urod, gata1:Cas9 vectors. |
Reprinted from Developmental Cell, 32(6), Ablain, J., Durand, E.M., Yang, S., Zhou, Y., Zon, L.I., A CRISPR/Cas9 Vector System for Tissue-Specific Gene Disruption in Zebrafish, 756-64, Copyright (2015) with permission from Elsevier. Full text @ Dev. Cell