RT Journal Article
SR Electronic
T1 Endogenous protein tagging in medaka using a simplified CRISPR/Cas9 knock-in approach
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2021.07.29.454295
DO 10.1101/2021.07.29.454295
A1 Ali Seleit
A1 Alexander Aulehla
A1 Alexandre Paix
YR 2021
UL http://biorxiv.org/content/early/2021/07/29/2021.07.29.454295.abstract
AB The CRISPR/Cas9 system has been used to generate fluorescently labelled fusion proteins by homology directed repair in a variety of species. Despite its revolutionary success, there remains an urgent need for increased simplicity and efficiency of genome editing in research organisms. Here, we establish a simplified, highly efficient and precise strategy for CRISPR/Cas9 mediated endogenous protein tagging in medaka (Oryzias latipes). We use a cloning-free approach that relies on PCR amplified donor fragments containing the fluorescent reporter sequences flanked by short homology arms (30-40bp), a synthetic sgRNA and streptavidin tagged Cas9. We generate six novel knock-in lines with high efficiency of F0 targeting and germline transmission. Whole Genome Sequencing (WGS) results reveal single-copy integration events only at the targeted loci. We provide an initial characterization of these fusion-protein lines, significantly expanding the repertoire of genetic tools available in medaka. In particular, we show that the mScarlet-pcna knock-in line has the potential to serve as an organismal-wide label for proliferative zones and an endogenous cell cycle reporter.