Efficient Single-Gene and Gene Family Editing in the Apicomplexan Parasite Eimeria tenella Using CRISPR-Cas9

Hu, Dandan; Tang, Xinming; Mamoun, Choukri Ben; Wang, Chaoyue; Wang, Si; Gu, Xiaorong; Duan, Chunhui; Zhang, Sixin; Suo, Jinxia; Deng, Miner; Yu, Yonglan; Suo, Xun; Liu, Xianyong

doi:10.3389/fbioe.2020.00128

Cited by 20 publications

(19 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Now we have achieved transgenic E. acervulina with a fluorescent rate of more than 95% by using only fluorescent protein (EYFP) as a screening marker (Yu et al, unpublished data). At the same time, our laboratory has also achieved CRISPR/Cas9 gene knockout on Eimeria [ 36 , 37 ]. Therefore, after obtaining a stable transgenic E. acervulina line, we can use a gene-editing system to knock out the other selection markers like YFP, RFP, and DHFR-TS.…”

Section: Discussionmentioning

confidence: 99%

Establishment of Recombinant Eimeria acervulina Expressing Multi-Copies M2e Derived from Avian Influenza Virus H9N2

et al. 2021

Self Cite

View full text Add to dashboard Cite

The potential of Eimeria parasites as live vaccine vectors has been reported with successful genetic manipulation on several species like E. tenella, E. mitis and E. necatrix. Among seven Eimeria species infecting chickens, E. acervulina is a highly prevalent, moderately pathogenic species. Thus, it is valuable for the study of transfection and for use as a potential as vaccine vector. In this study, a plasmid containing expression cassette with enhanced yellow fluorescent protein (EYFP), red fluorescent protein (RFP) and 12 copies of extracellular domain of H9N2 avian influenza virus M2 (M2e) protein was used for the transfection. Nucleofected sporozoites were inoculated into birds through wing vein. Recombinant E. acervulina oocysts with 0.1% EYFP+ and RFP+ populations were collected from the feces of the inoculated birds. The fluorescent rate of transgenic parasites reached over 95% after nine successive propagations with a pyrimethamine selection in vivo and fluorescent-activated cell sorting (FACS) of progeny oocysts. The expression of M2e in the transgenic parasites (EaM2e) was confirmed by Western blot and its cytoplasm localization in sporozoites was displayed by an indirect immunofluorescent assay (IFA). Meanwhile, we found that the fecundity of EaM2e was equivalent to that of wild type E. acervulina (EaWT). Taken together, the stable transfection of E. acervulina was successfully established. Future studies will focus on whether transgenic E. acervulina can serve as a live vaccine vector.

show abstract

Section: Discussionmentioning

confidence: 99%

Establishment of Recombinant Eimeria acervulina Expressing Multi-Copies M2e Derived from Avian Influenza Virus H9N2

et al. 2021

Self Cite

View full text Add to dashboard Cite

show abstract

“…The programs EO100, EH100, and EO114 in Lonza 4D-Nucleofector have higher transfection efficiency than others ( Supplementary Figure S6 ). So far, there are two reported genome editing studies ( Hu et al, 2020 ; Tang et al, 2020 ). We summarized it as Supplementary Table S3 .…”

Section: Discussionmentioning

confidence: 99%

“…Several studies have been performed for optimization of the transgenic promoters, fluorescent reporter genes, fusion strategy, and selection for enrichment of positive transformants ( Clark et al, 2008 ; Hanig et al, 2012 ; Tang et al, 2016 ; Pastor-Fernandez et al, 2019 ), i.e., using the selection marker dihydrofolate reductase–thymidylate synthase ( DHFR ) gene ( Liu et al, 2008 ). Due to the lack of a method providing a continuous culture of Eimeria at one stage as Toxoplasma , no genome editing techniques have been reported until the past year ( Hu et al, 2020 ; Tang et al, 2020 ). It was reported that an E. tenella parasite strain harboring eCas9 expression would facilitate gene manipulation.…”

Section: Introductionmentioning

confidence: 99%

“…It was reported that an E. tenella parasite strain harboring eCas9 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/Cas9 plasmid system successfully tagged the endogenous microneme protein 2 ( EtMic2 ) with a red fluorescent protein ( Tang et al, 2020 ).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

FnCas12a/crRNA-Mediated Genome Editing in Eimeria tenella

et al. 2021

View full text Add to dashboard Cite

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.

show abstract

“…Transcriptional analysis of the pre-sexual stages of E. tenella revealed that ApiAP2 expression profile changes during pre-sexual development ( Su et al., 2017 ). Individual deletion of 10 out of 33 ApiAP2 factors was successful in a screening performed in sporozoites of E. tenella ( Hu et al., 2020 ). This finding indicates that these 10 ApiAP2 are likely dispensable in E. tenella .…”

Section: Sexual Commitment and Differentiation In Coccidiamentioning

confidence: 99%

Comparisons of the Sexual Cycles for the Coccidian Parasites Eimeria and Toxoplasma

Genova

Knoll

2020

Front. Cell. Infect. Microbiol.

View full text Add to dashboard Cite

Toxoplasma gondii and Eimeria spp. are widely prevalent Coccidian parasites that undergo sexual reproduction during their life cycle. T. gondii can infect any warm-blooded animal in its asexual cycle; however, its sexual cycle is restricted to felines. Eimeria spp. are usually restricted to one host species, and their whole life cycle is completed within this same host. The literature reviewed in this article comprises the recent findings regarding the unique biology of the sexual development of T. gondii and Eimeria spp. The molecular basis of sex in these pathogens has been significantly unraveled by new findings in parasite differentiation along with transcriptional analysis of T. gondii and Eimeria spp. pre-sexual and sexual stages. Focusing on the metabolic networks, analysis of these transcriptome datasets shows enrichment for several different metabolic pathways. Transcripts for glycolysis enzymes are consistently more abundant in T. gondii cat infection stages than the asexual tachyzoite stage and Eimeria spp. merozoite and gamete stages compared to sporozoites. Recent breakthroughs in host-pathogen interaction and host restriction have significantly expanded the understating of the unique biology of these pathogens. This review aims to critically explore advances in the sexual cycle of Coccidia parasites with the ultimate goal of comparing and analyzing the sexual cycle of Eimeria spp. and T. gondii.

show abstract

Efficient Single-Gene and Gene Family Editing in the Apicomplexan Parasite Eimeria tenella Using CRISPR-Cas9

Cited by 20 publications

References 41 publications

Establishment of Recombinant Eimeria acervulina Expressing Multi-Copies M2e Derived from Avian Influenza Virus H9N2

Establishment of Recombinant Eimeria acervulina Expressing Multi-Copies M2e Derived from Avian Influenza Virus H9N2

FnCas12a/crRNA-Mediated Genome Editing in Eimeria tenella

Comparisons of the Sexual Cycles for the Coccidian Parasites Eimeria and Toxoplasma

Contact Info

Product

Resources

About