Detection of CRISPR-Cas-induced mutations in Daphnia.

CRISPR-Cas9 has established itself as a robust tool for conducting loss-of-function gene research in emerging model species, including the freshwater zooplankton Daphnia. However, sensitive detection of mutations, especially in genetic mosaic and pooled samples, remains a challenge. In this study, we evaluate 2 of the most widely used mutation screening techniques, the T7 Endonuclease I assay and fragment analysis for their sensitivity, accuracy, and practical use in detecting CRISPR-induced indels in 4 targeted genes, DNMT3A, DNMT3B, PERIOD2, and DMRT1 in Daphnia magna. Here, we show that T7 Endonuclease I, although it offers a quick and cost-effective screening method, often produces false positives, especially when examining pooled samples. Conversely, fragment analysis facilitates detecting allele size differences at a fine resolution, reproducibility in detecting indels, and distinguishing zygosity, and is more reliable as a method to detect mutation. Our comparative analyses convey the importance of carefully selecting the appropriate screening methods depending on research questions.