Abstract
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.
Highlights
Genomes are threatened by endogenously generated chemicals like reactive oxygen species and exogenous compounds such as mutagenic agents and radiation[1], which can lead to DNA double-strand breaks (DSBs)
The 10 injected embryos survived until adult, from which 9 produced offspring with a white eye that is the typical phenotype of the scarlet mutant, indicating that the Cas[9] RNP induced DSBs at the targeted site
We evaluated the functionality of this reporter system by introducing targeted DSB in the reporter site
Summary
Genomes are threatened by endogenously generated chemicals like reactive oxygen species and exogenous compounds such as mutagenic agents and radiation[1], which can lead to DNA double-strand breaks (DSBs). The sequenced genome of Daphnia reveals highly duplicated genes, resulting in tandem gene c lusters[13] These tandem clusters may serve as a template for HDR-based repair to attenuate the effect of deleterious mutations during the parthenogenetic cycle, which suggests that Daphnia may have a unique HDR mechanism. The effects of genotoxicants have been investigated at the phenotypic level[14,15] To understand their actions at the molecular level, it is important to study the DNA repair mechanism in this species. The HDR-based knock-in efficiency was low probably due to competition with the NHEJ pathway To test this hypothesis, disruption DNA ligase IV which is the conserved component of the NHEJ pathway has been attempted[16] its effect was not fully evaluated due to the lack of a method for quantifying the HDR event in vivo. A system to evaluate and quantify the HDR event is a necessity
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