Fig. 2

Fig. 2

Integratable CRISPR Vector for Tissue-Specific Gene Targeting

(A) Schematic representation of the tissue-specific CRISPR vector. Prom denotes any promoter of interest used to drive Cas9 expression in a tissue-restricted manner. GFP expression in the heart of injected embryos is used as a transgenesis marker. Tol2 indicates transposition sites for the Tol2 transposase, SV40 polyA sequence (pA). See Figure S2A for the sequence of U6:gRNA, which comprises two BseRI restriction sites allowing easy cloning of any gene-specific target sequence at the 5′ end of the gRNA.

(B) Outline of the experiment, the CRISPR vector is injected into one-cell stage embryos along with Tol2 mRNA. Embryos with GFP-positive hearts are sorted at 24 hpf and further analyzed. Cas9 expression is assessed by in situ hybridization of Cas9 mRNA, targeted mutation efficiency by T7E1 assay and sequencing, and the presence of fluorescent red blood cells by confocal microscopy.

(C) Confocal images of embryos injected with Tol2 mRNA and the pcmlc2:GFP, U6:GFP, gata1:Cas9 vector at various time points. Note early (4 hpf) and ubiquitous (24 hpf) GFP expression.

(D) Representative images show whole mount in situ hybridization (WISH) using an anti-sense RNA probe against Cas9 mRNA in 24 hpf embryos injected with Tol2 mRNA and pcmlc2:GFP, U6:gRNA urod vectors expressing Cas9 under the control of the indicated promoters. Cas9 expression pattern is governed by the tissue-specificity of the promoters. See also Figure S2B.