Abnormal expression pattern of knock-in marker in Eimeria tenella using CRISPR/Cas9

Gene editing technology has been widely applied in the genetic manipulation of many organisms and is increasingly being utilized in eukaryotic pathogens. However, its efficiency often requires improvement. In our study using CRISPR/Cas9 to genetically manipulate Eimeria tenella, we aimed to insert a tag into a target gene locus via homologous recombination, but observed outcomes inconsistent with expectations. Whole-genome sequencing analysis of the integration sites revealed that the transgenic E. tenella did not exhibit correct targeted integration. These results indicate that creating double-strand breaks (DSB) at specific genomic sites to trigger homology-directed repair (HDR) for gene modification can lead to mislocalized expression. This study provides insights for utilizing CRISPR/Cas9 technology in genetic editing, particularly in E. tenella, and offers suggestions for improving strategies that employ the co-transfection of multiple plasmids, such as Cas9-gRNA and donor plasmids.