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

Area of Science:

• Molecular Biology
• Parasitology
• Genetics

Background:

• Gene editing technologies, including CRISPR/Cas9, are vital for genetic manipulation in organisms.
• Their application in eukaryotic pathogens like Eimeria tenella is growing, but efficiency needs enhancement.
• CRISPR/Cas9 facilitates targeted DNA modifications through double-strand breaks (DSBs) and homology-directed repair (HDR).

Purpose of the Study:

• To genetically manipulate Eimeria tenella using CRISPR/Cas9.
• To insert a tag into a target gene locus via homologous recombination.
• To investigate the efficiency and accuracy of CRISPR/Cas9-mediated gene editing in this pathogen.

Main Methods:

• Utilized CRISPR/Cas9 technology for genetic manipulation in Eimeria tenella.
• Aimed for homologous recombination to insert a tag into a specific gene locus.
• Performed whole-genome sequencing to analyze integration sites in transgenic parasites.

Main Results:

• Observed outcomes inconsistent with the intended targeted integration.
• Whole-genome sequencing revealed that integration occurred at unintended sites.
• Transgenic Eimeria tenella did not show correct targeted integration as expected.

Conclusions:

• CRISPR/Cas9-induced double-strand breaks (DSBs) can lead to mislocalized expression, not precise homology-directed repair (HDR).
• This study provides critical insights into CRISPR/Cas9 gene editing in Eimeria tenella.
• Suggests improved strategies for co-transfection of multiple plasmids (Cas9-gRNA and donor) for enhanced gene editing efficiency.