CRISPR/Cas9-mediated knock-in of KalTA4 into the Tg(neurod:eGFP) transgenic line. (A) A schematic of the donor plasmid consisting of an N-terminal eGFPbait with two sgRNA target sites (in orange, PAM sequence in blue). After co-injection of the donor with Cas9 mRNA and one eGFP sgRNA, insertion at the eGFP locus occurs. In-frame fusion of the E2A-KalTA4-pA cassette results in a multicistronic mRNA after successful integration at the eGFP locus. Due to the E2A sequence, the N-terminal eGFP peptide is cleaved from the KalTA4 protein by cotranslational ribosomal skipping. (B) A 6-dpf Tg(neurod:eGFP) × Tg(UAS:RFP, cry1:eGFP) embryo showing a switch from eGFP- to RFP-expressing cells upon injection of the donor plasmid together with sgRNA eGFP 1 and Cas9 mRNA. Successful in-frame knock-in of the donor plasmid into the eGFP open reading frame results in KalTA4 expression. Consecutively, KalTA4 binds to UAS:RFP and triggers RFP expression, leading to the eGFP to RFP switch. Scale bar, 300 μm. Tg(UAS:RFP, cry1:eGFP) transgenic fish express eGFP in the lens (driven by the crystalline promoter cry1:eGFP), thus allowing UAS:RFP transgenic fish to be identified by expression of eGFP in their lens (since without transactivation by KalTA4, no RFP is expressed from this transgene). (C) No RFP-expressing cells could be observed in Tg(neurod:eGFP) × Tg(UAS:RFP, cry1:eGFP) embryos injected with the donor plasmid and Cas9 mRNA but without sgRNA eGFP 1. Scale bar, 300 μm. (D) A representative gel of PCR products obtained from the founder fish shown in B, demonstrating targeted knock-in of the donor plasmid at the eGFP locus. PCR primers were placed flanking the neurod:eGFP locus and outward directed in the donor plasmid. Positions of PCR primers and the resulting fragment nomenclature are shown in A. (E) Sequence analysis at the 5′ and 3′ junctions of five representative targeted integration events. (Orange) sgRNA binding site, (red) base pair changes or insertions. The PAM sequence NGG required for cleavage by Cas9 (Jinek et al. 2012) is shown in blue. Note that only the Δ6 integration events correspond to in-frame insertions of the E2A-KalTA4 sequence. Due to three possible frames and two integration directions, only 16.6% of integration events will result in RFP expression.
