ArticlePloS one2017
CRISPR/Cas-mediated knock-in via non-homologous end-joining in the crustacean Daphnia magna.
Article in PloS one, 2017. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 26 papers.
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Who cites it
26 citing papers in PubMed.
- Establishment of a CRISPR/Cas9-Based Genome Editing System inAnimals : an open access journal from MDPI · 2025Article
- Article
- Baculovirus and Plasmid Vector-Mediated Gene Transfer inBio-protocol · 2025Article
- The mosaicism of Cas-induced mutations and pleiotropic effects of scarlet gene in an emerging model system.Heredity · 2025Article
- Article
- Maternal effects in the model system Daphnia: the ecological past meets the epigenetic future.Heredity · 2025Review
- Spatiotemporal control of transgene expression using an infrared laser in the crustacean Daphnia magna.Scientific reports · 2024Article
- Knockout of a single Pax6 gene (toy but not ey) leads to compound eye deficiency and small head in honeybees.Communications biology · 2024Article
- Daphnia sp. (Branchiopoda: Cladocera) Mitochondrial Genome Gene Rearrangement and Phylogenetic Position Within Branchiopoda.Biochemical genetics · 2024Article
- Identification of gene isoforms and their switching events between male and female embryos of the parthenogenetic crustacean Daphnia magna.Scientific reports · 2024Article
- Efficient CRISPR genome editing and integrative genomic analyses reveal the mosaicism of Cas-induced mutations and pleiotropic effects ofbioRxiv : the preprint server for biology · 2024Article
- Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR-Associated Protein and Its Utility All at Sea: Status, Challenges, and Prospects.Microorganisms · 2024Review
- Marine-montane transitions coupled with gill and genetic convergence in extant crustacean.Science advances · 2023Article
- QTL study reveals candidate genes underlying host resistance in a Red Queen model system.PLoS genetics · 2023Article
- CRISPR/Cas9 establishment-mediated targeted mutagenesis inFrontiers in physiology · 2023Article
- Review
- Regulation of Doublesex1 Expression for Environmental Sex Determination in the Cladoceran Crustacean Daphnia.Frontiers in cell and developmental biology · 2022Review
- Genomic Signatures of Local Adaptation in Clam Shrimp (Eulimnadia texana) from Natural Vernal Pools.Genome biology and evolution · 2020Article
- Experimental duplication of bilaterian body axes in spider embryos: Holm's organizer and self-regulation of embryonic fields.Development genes and evolution · 2020Review
- Delivery methods for CRISPR/Cas9 gene editing in crustaceans.Marine life science & technology · 2020Article
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Authors and funding
5 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system (Cas) is widely used for mediating the knock-in of foreign DNA into the genomes of various organisms. Here, we report a process of CRISPR/Cas-mediated knock-in via non-homologous end joining by the direct injection of Cas9/gRNA ribonucleoproteins (RNPs) in the crustacean Daphnia magna, which is a model organism for studies on toxicology, ecology, and evolution. First, we confirmed the cleavage activity of Cas9 RNPs comprising purified Cas9 proteins and gRNAs in D. magna. We used a gRNA that targets exon 10 of the eyeless gene. Cas9 proteins were incubated with the gRNAs and the resulting Cas9 RNPs were injected into D. magna eggs, which led to a typical phenotype of the eyeless mutant, i.e., eye deformity. The somatic and heritable mutagenesis efficiencies were up to 96% and 40%, respectively. Second, we tested the CRISPR/Cas-mediated knock-in of a plasmid by the injection of Cas9 RNPs. The donor DNA plasmid harboring the fluorescent reporter gene was designed to contain the gRNA recognition site. The co-injection of Cas9 RNPs together with the donor DNAs resulted in generation of one founder animal that produced fluorescent progenies. This transgenic Daphnia had donor DNA at the targeted genomic site, which suggested the concurrent cleavage of the injected plasmid DNA and genomic DNA. Owing to its simplicity and ease of experimental design, we suggest that the CRISPR/Cas-mediated knock-in method represents a promising tool for studying functional genomics in D. magna.
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