FnCas12a/crRNA-Mediated Genome Editing in Eimeria tenella

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

doi:10.3389/fgene.2021.738746

Abstract

Eimeria 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 Eimeria is of immense importance for the development of vaccines and drugs. CRISPR/Cas9 has been utilized for manipulating the genome of Eimeria tenella (E. tenella). Ectopic expression of Cas9, i.e., via 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 EtHistone H4 triggered DNA double-strand breaks in vivo. We then used this strategy to knock-in a coding cassette for an enhanced yellow fluorescent protein (EYFP) and dihydrofolate reductase–thymidylate synthase gene (DHFR) as a selection marker to tag endogenous EtActin. The engineered E. tenella parasite possesses EYFP expression in its entire life cycle. Our results demonstrated that FnCas12a could trigger genome editing in E. tenella, which augments the applicability of the dissection of gene function and the development of anticoccidial drugs and vaccines for Eimeria species.

Highlights

Coccidiosis is one of the most important animal parasitic diseases
These results demonstrated that FnCas12a–RNP could be used for manipulating the E. tenella genome
We evaluated whether FnCas12a could be utilized for the manipulation of the E. tenella genome

Summary

INTRODUCTION

Coccidiosis is one of the most important animal parasitic diseases. It is reported in several species, including chickens, ducks, puppies, rabbits, piglets, dogs, horses, kittens, and birds, and is caused by the members of the Eimeria genus (Stock et al, 2018). It was reported that an E. tenella parasite strain harboring eCas expression would facilitate gene manipulation Using this specific strain, the plasmid for sgRNA expression plus the donor fragment would trigger genome editing (Hu et al, 2020). The other study used the CRISPR/ Cas plasmid system successfully tagged the endogenous microneme protein 2 (EtMic2) with a red fluorescent protein (Tang et al, 2020) These advances promoted the functional genomics study of Eimeria species. These results demonstrated that FnCas12a–RNP could be used for manipulating the E. tenella genome This technique would significantly augment the development of vaccines and drugs against Eimeria species as well as the dissection of novel gene functions

MATERIALS AND METHODS

RESULTS

DISCUSSION

DATA AVAILABILITY STATEMENT