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

Abstract

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 (Oryzias latipes). 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.