Phenotype and gene-editing evaluation of Cas9-GFP expressing daphnia magna

• Main Author: Lumio, Rob T.
• Format: text
• Language: English
• Published: Animo Repository 2024
• Subjects: Daphnia magna; CRISPR-associated protein 9; fluorescence microscopy; Gene editing; Chemistry
• Online Access: https://animorepository.dlsu.edu.ph/etdm_chem/21; https://animorepository.dlsu.edu.ph/context/etdm_chem/article/1021/viewcontent/2024_Lumio_Phenotype_and_Gene_editing_Evaluation_of_Cas9_GFP_Expressing_Daph.pdf
• Institution: De La Salle University

Daphnia magna is a valuable model organism in environmental toxicology, but efficient gene-editing tools are crucial for elucidating gene function. This study evaluated a novel Cas9-GFP expressing D. magna strain for CRISPR-based gene editing. We assessed the transgenic phenotype by comparing growth rates and fecundity with wild-type strain, finding minimal impact on overall fitness. Cas9 expression was confirmed in embryos at 9 hours post-development. To optimize gene editing efficiency, we targeted the scarlet gene (DapmaST), responsible for eye pigmentation, using both capped and 2'-O-methylated gRNAs. While capped gRNA resulted in partial loss of eye pigmentation, methylated gRNA achieved complete loss, demonstrating superior editing efficacy. This study validates the functionality of our Cas9-GFP expressing D. magna model and highlights the importance of gRNA modification in enhancing CRISPR efficiency. Our findings establish an improved system for loss-of-function studies in D. magna, advancing its utility in environmental and genetic research.