AUTHOR=Cheng Peipei , Zhang Zhihao , Yang Fayu , Cai Shuo , Wang Lina , Wang Chunmei , Wang Mi , Liu Yingchun , Fei Chenzhong , Zhang Lifang , Xue Feiqun , Gu Feng 

TITLE=FnCas12a/crRNA-Mediated Genome Editing in Eimeria tenella

JOURNAL=Frontiers in Genetics

VOLUME=12

YEAR=2021

URL=https://www.frontiersin.org/journals/genetics/articles/10.3389/fgene.2021.738746

DOI=10.3389/fgene.2021.738746

ISSN=1664-8021

ABSTRACT=<p><italic>Eimeria</italic> species are intracellular parasites residing inside the intestinal epithelial cell, which cause poultry coccidiosis and result in significant financial losses in the poultry industry. Genome editing of <italic>Eimeria</italic> is of immense importance for the development of vaccines and drugs. CRISPR/Cas9 has been utilized for manipulating the genome of <italic>Eimeria tenella</italic> (<italic>E. tenella</italic>). Ectopic expression of Cas9, i.e., <italic>via</italic> plasmids, would introduce transgene, which substantially limits its application, especially for vaccine development. In this study, we initially optimized the condition of the transfection protocol. We demonstrated that with the optimized condition, the transfection of FnCas12a (also known as “FnCpf1”) protein and crRNA targeting <italic>EtHistone H4</italic> triggered DNA double-strand breaks <italic>in vivo</italic>. We then used this strategy to knock-in a coding cassette for an enhanced yellow fluorescent protein (<italic>EYFP</italic>) and dihydrofolate reductase–thymidylate synthase gene (<italic>DHFR</italic>) as a selection marker to tag endogenous <italic>EtActin</italic>. The engineered <italic>E. tenella</italic> parasite possesses <italic>EYFP</italic> expression in its entire life cycle. Our results demonstrated that FnCas12a could trigger genome editing in <italic>E. tenella</italic>, which augments the applicability of the dissection of gene function and the development of anticoccidial drugs and vaccines for <italic>Eimeria</italic> species.</p>