Fig. 1

An overview of the TICIT technology for visual genotyping, promoter-tagging, and single-copy, site-specific transgenesis. CRISPR-Cas9 from S. pyogenes (SpCas9) is employed together with a single-stranded oligodeoxynucleotide (ssODN) containing the phiC31 landing site to create attP knock-in fish lines. Next, single-copy site-specific transgene integration is achieved by co-delivering the phiC31 mRNA with any plasmids containing the phiC31 attachment sequence attB. For visual genotyping, two plasmids carrying fluorescent markers driven by a promoter of user's choice (shown as unshaded boxes) are used. This enables Cas9-mediated gene mutations to be identified by fluorescence even before mutant phenotypes manifest. In a heterozygous intercross, wild-type offspring are unmarked, heterozygous offspring are either green or red, and homozygous offspring are both green and red (shown as yellow). For promoter-tagging, a plasmid carrying a promoter-less fluorescent marker adjacent to the attB sequence is used. After phiC31-mediated integration, the promoter activity of the target gene can be tracked by the expression of the fluorescent marker. Further, fish lines carrying attP knock-in at pre-defined loci may enable facile transgenesis devoid of positional and copy number artifacts.