Instantaneous visual genotyping and facile site-specific transgenesis via CRISPR-Cas9 and phiC31 integrase

The zebrafish Danio rerio has become a popular model in functional genomics and genetic disease studies. However, when a zebrafish mutant line must be propagated as heterozygotes due to homozygous lethality, using standard genotyping methods to identify a population of homozygous mutant embryos is time-consuming and sometimes impractical due to downstream applications such as large-scale chemical screens. Here, we introduce TICIT, Targeted Integration by CRISPR-Cas9 and Integrase Technologies, which utilizes the site-specific DNA recombinase - phiC31 integrase - to insert fluorescent markers into CRISPR-Cas9-generated mutant alleles. It allows instantaneous determination of the genotype of a zebrafish simply by examining its color. This technique, which relies on first knocking in a 39-basepair phiC31 landing site via CRISPR-Cas9, enables researchers to insert large DNA fragments at the same genomic location repeatedly and with high precision and efficiency. We demonstrated that TICIT could also be used to create reporter fish driven by an endogenous promoter. Additionally, we created a landing site located in the tyrosinase gene that could support transgene expression in a broad spectrum of tissue and cell types, acting as a putative safe harbor locus. Hence, TICIT can yield predictable and reproducible transgene expression, facilitate diverse applications in zebrafish, and may be applicable to cells in culture and other model organisms. ### Competing Interest Statement The authors have declared no competing interest.