ArticleBio-protocol2025
Cloning-Free Targeting of Endogenous Loci to Generate Fluorescent Reporters in Medaka.
Article in Bio-protocol, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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4 authors.
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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 (
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