Genetic modification of the protozoan Eimeria tenella using the CRISPR/Cas9 system

Tang, Xinming; Suo, Jingxia; Liang, Lin; Duan, Chunhui; Hu, Dandan; Gu, Xiaorong; Yu, Yonglan; Liu, Xianyong; Cui, Shangjin; Suo, Xun

doi:10.1186/s13567-020-00766-0

Cited by 15 publications

(12 citation statements)

References 23 publications

(31 reference statements)

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“…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with a fluorescent rate of more than 95% by using only fluorescent protein (EYFP) as a screening marker (Yu et al, unpublished data).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

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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.

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Section: Discussionmentioning

confidence: 99%

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

et al. 2021

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“…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%

“…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 ). These advances promoted the functional genomics study of Eimeria species.…”

Section: Introductionmentioning

confidence: 99%

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FnCas12a/crRNA-Mediated Genome Editing in Eimeria tenella

et al. 2021

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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.

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“…It will be needed to generate mutated versions of the Eimeria P4022-containing proteins targeting the P4022 sequence by genetically modifying Eimeria using the CRISPR/Cas9 system. [101] 2. RNAseq approach.…”

Section: Proposed Experiments To the Polyq/cxcr-type Receptor And The Lamina Propria Invasion Modelmentioning

confidence: 99%

From celiac disease to coccidia infection and vice‐versa: The polyQ peptide CXCR3‐interaction axis

et al. 2021

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Zonulin is a physiological modulator of intercellular tight junctions, which upregulation is involved in several diseases like celiac disease (CeD). The polyQ gliadin fragment binds to the CXCR3 chemokine receptor that activates zonulin upregulation, leading to increased intestinal permeability in humans. Here, we report a general hypothesis based on the structural connection between the polyQ sequence of the immunogenic CeD protein, gliadin, and enteric coccidian parasites proteins. Firstly, a novel interaction pathway between the parasites and the host is described based on the structural similarities between polyQ gliadin fragments and the parasite proteins. Secondly, a potential connection between coccidial infections as a novel environmental trigger of CeD is hypothesized. Therefore, this report represents a promising breakthrough for coccidian research and points out the potential role of coccidian parasites as a novel trigger of CeD that might define a preventive strategy for gluten-related disorders in general. Also see the video abstract here: https://youtu.be/oMaQasStcFI

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Genetic modification of the protozoan Eimeria tenella using the CRISPR/Cas9 system

Cited by 15 publications

References 23 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

From celiac disease to coccidia infection and vice‐versa: The polyQ peptide CXCR3‐interaction axis

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