Endogenous protein tagging in medaka using a simplified CRISPR/Cas9 knock-in approach

Abstract
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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)

The following data sets were generated

(2021) WGS on CRISPR mediated Knock-ins in medaka
European Nucleotide Archive, PRJEB43219.

https://www.ebi.ac.uk/ena/browser/view/PRJEB43219?show=reads

Article and author information

Author details

Ali Seleit

Developmental Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany

For correspondence
ali.seleit@embl.de

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-8144-2286
Alexander Aulehla

Developmental Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0003-3487-9239
Alexandre Paix

Developmental Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany

For correspondence
alexandre.paix@embl.de

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-8080-7546

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

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.