Endogenous protein tagging in medaka using a simplified CRISPR/Cas9 knock-in approach
- European Molecular Biology Laboratory, Germany
Abstract
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 Cas9 mRNA. We generate eight 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 line has the potential to serve as an organismal-wide label for proliferative zones and an endogenous cell cycle reporter.
Data availability
Sequencing data have been deposited in European Nucleotide Archive (ENA) under study number ERP127162. Accession numbers are: eGFP-cbx1b(1) ERS5796960 (SAMEA8109891), eGFP-cbx1b(2) ERS5796961 (SAMEA8109892), mScarlet-pcna ERS5796962 (SAMEA8109893) and mNeonGreen-myosinhc ERS5796963 (SAMEA8109894)
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WGS on CRISPR mediated Knock-ins in medakaEuropean Nucleotide Archive, PRJEB43219.
Article and author information
Author details
Funding
H2020 European Research Council (866537)
- Ali Seleit
EMBL interdisciplinary Postdoc (847543)
- Ali Seleit
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: Medaka (Oryzias latipes, Cab strain) (Iwamatsu, 2004, Naruse et al., 2004, Kasahara et al., 2007) were maintained as closed stocks in a fish facility built according to the European Union animal welfare standards and all animal experiments were performed in accordance with European Union animal welfare guidelines. Animal experimentation was approved by The EMBL Institutional Animal Care and Use Committee (IACUC) project code: 20/001_HD_AA. Fishes were maintained in a constant recirculating system at 27-28{degree sign}C with a 14hr light /10hr dark cycle.
Copyright
© 2021, Seleit et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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