Cloning-Free Targeting of Endogenous Loci to Generate Fluorescent Reporters in Medaka.

CRISPR-Cas9 has democratized genome engineering due to its simplicity and efficacy. Adapted from a bacterial defense mechanism, CRISPR-Cas9 comprises the Cas9 endonuclease and a site-specific guide RNA. In vivo, the Cas9 ribonucleoprotein (RNP) can target specific genomic loci and generate double-strand breaks. Eukaryotic endogenous DNA repair mechanisms recognize the cut site and attempt to repair the DNA either by non-homologous end joining, which introduces insertions/deletions, resulting in a loss of reading frame in coding genes, or through homology-directed repair that maintains the reading frame. The latter approach allows the insertion of fluorescent reporter sequences in frame with protein-coding genes in order to monitor gene expression and protein dynamics in cells and whole organisms. Here, we provide a protocol for targeting endogenous genes to introduce sequences coding for fluorescent reporters in medaka (). The method is simple, robust, and efficient, thus facilitating straightforward organismal genome editing. Key features • Cloning free CRISPR/Cas9 tagging of endogenous genes with fluorescent reporter sequences. • Guidelines for designing CRISPR/Cas9 endogenous tagging experiments. • Straightforward generation of transgenic Medaka knock-in reporter lines. • Versatility with the use of Cas9 mRNA or protein and various fluorescent reporters.