Development of transgenic Daphnia magna for visualizing homology-directed repair of DNA

• Published in: Scientific reports Vol. 12; no. 1; pp. 2497 - 12
• Main Authors: Fatimah, Rizky Mutiara, Adhitama, Nikko, Kato, Yasuhiko, Watanabe, Hajime
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 15-02-2022; Nature Publishing Group; Nature Portfolio
• Subjects: 631/1647/334/1582; 631/337/1427/2190; Animals; Animals, Genetically Modified - genetics; CRISPR; CRISPR-Cas Systems; Crustaceans; Daphnia - genetics; Daphnia magna; DNA - genetics; DNA End-Joining Repair - genetics; DNA repair; Environmental effects; Fluorescence; Gene Editing - methods; Gene Knock-In Techniques; Genes, Reporter; Genome editing; Genomes; Genotype; Green fluorescent protein; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Homology; Humanities and Social Sciences; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; multidisciplinary; Parthenogenesis; Plasmids; Polymerase chain reaction; Promoter Regions, Genetic - genetics; Recombinational DNA Repair - genetics; Red Fluorescent Protein; Reporter gene; Science; Science (multidisciplinary); Signal Transduction - genetics; Toxicants
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-022-06526-8

In the crustacean
Daphnia magna
, studying homology-directed repair (HDR) is important to understand genome maintenance during parthenogenesis, effects of environmental toxicants on the genome, and improvement of HDR-mediated genome editing. Here we developed a transgenic
D. magna
that expresses green fluorescence protein (GFP) upon HDR occurrence. We utilized the previously established reporter plasmid named DR-GFP that has a mutated
eGFP
gene (
SceGFP
) and the tandemly located donor
GFP
gene fragment (
iGFP
). Upon double-strand break (DSB) introduction on
SceGFP
, the
iGFP
gene fragment acts as the HDR template and restores functional eGFP expression. We customized this reporter plasmid to allow bicistronic expression of the
mCherry
gene under the control of the
D. magna EF1α-1
promoter/enhancer. By CRISPR/Cas-mediated knock-in of this plasmid via non-homologous joining, we generated the transgenic
D. magna
that expresses mCherry ubiquitously, suggesting that the DR-GFP reporter gene is expressed in most cells. Introducing DSB on the
SceGFP
resulted in eGFP expression and this HDR event could be detected by fluorescence, genomic PCR, and quantitative reverse-transcription PCR, suggesting this line could be used for evaluating HDR. The established reporter line might expand our understanding of the HDR mechanism and also improve the HDR-based gene-editing system in this species
.