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

Ali Seleit,Alexander Aulehla,Alexandre Paix

doi:10.7554/elife.75050

Ali Seleit, Alexander Aulehla + Show 1 more

Open Access

https://doi.org/10.7554/elife.75050

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Journal: eLife	Publication Date: Dec 6, 2021
Citations: 20	License type: CC BY 4.0

Affiliation: European Molecular Biology Laboratory

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 (30-40 bp), a synthetic single-guide RNA and Cas9 mRNA. We generate eight novel knock-in lines with high efficiency of F0 targeting and germline transmission. Whole genome sequencing 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.

Highlights

86 we provide proof of principle evidence that the endogenous mScarlet87 pcna knock-in we generate serves as a bona fide proliferative cell label and an endogenous cell cycle reporter, with broad application potential in a vertebrate model system
Lastly, we reveal that proliferative cells are present in the spinal cord of stage 40 medaka embryos, a finding that has not been previously reported, and we show that these mScarlet-Pcna positive cells occur in clusters preferentially located on the dorsal side of the spine (Figure 3- figure supplement 1, n= 4 embryos)
In medaka there are a total of three validated single447 copy fusion protein lines by CRISPR/CRISPR associated protein 9 (Cas9) reported prior to this work (Gutierrez-Triana et al, 2018), and in zebrafish, a handful of lines have been reported so far

Summary

Introduction

The advent of gene editing tools (Wang et al, 2016, Jinek et al, 2012, Cong et al., 2013) in conjunction with the expansion of sequenced genomes and engineered fluorescent proteins (Chudakov et al, 2010, Shaner et al, 2013, Bindels et al, 2017, Campbell et al, 2020) has revolutionized the ability to generate endogenous fusion protein Knock-In (KI) lines in a growing number of organisms (Paix et al, 2015, Paix et al, 2017a, Gratz et al, 2014, Kanca et al, 2019, Wierson et al, 2020, Gutierrez-Triana et al, 2018, Auer and Del Bene, 2014, Yoshimi et al, 2016, Yao et al, 2017, Cong et al, 2013, Dickinson et al, 2015, Leonetti et al, 2016, Wierson et al, 2019) These molecular markers expressed at physiological levels are central to our understanding of cellular and tissue level dynamics during embryonic development (Gibson et al, 2013). Repair donors with shorter homology arms in combination with in vivo linearization of the donor plasmid have been shown to mediate efficient Knock-Ins in zebrafish and in mammalian cells (Wierson et al., 2020, Hisano et al, 2015, Cristea et al, 2013, Yao et al, 2017)

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