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Genetic manipulation of giant viruses and their host, Acanthamoeba castellanii

Nature Protocols volume 19, pages 3–29 (2024)Cite this article

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Abstract

Giant viruses (GVs) provide an unprecedented source of genetic innovation in the viral world and are thus, besides their importance in basic and environmental virology, in the spotlight for bioengineering advances. Their host, Acanthamoeba castellanii, is an accidental human pathogen that acts as a natural host and environmental reservoir of other human pathogens. Tools for genetic manipulation of viruses and host were lacking. Here, we provide a detailed method for genetic manipulation of A. castellanii and the GVs it plays host to by using CRISPR–Cas9 or homologous recombination. We detail the steps of vector preparation (4 d), transfection of amoeba cells (1 h), infection (1 h), selection (5 d for viruses, 2 weeks for amoebas) and cloning of recombinant viruses (4 d) or amoebas (2 weeks). This procedure takes ~3 weeks or 1 month for the generation of recombinant viruses or amoebas, respectively. This methodology allows the generation of stable gene modifications, which was not possible by using RNA silencing, the only previously available reverse genetic tool. We also include detailed sample-preparation steps for protein localization by immunofluorescence (4 h), western blotting (4 h), quantification of viral particles by optical density (15 min), calculation of viral lethal dose 50 (7 d) and quantification of DNA replication by quantitative PCR (4 h) to allow efficient broad phenotyping of recombinant organisms. This methodology allows the function of thousands of ORFan genes present in GVs, as well as the complex pathogen-host, pathogen-pathogen or pathogen-symbiont interactions in A. castellanii, to be studied in vivo.

Key points

  • This protocol enables genetic manipulation of nuclear and cytoplasmic giant viruses and their host, Acanthomoeba castellanii, by using either CRISPR–Cas9 or homologous recombination.

  • The methodology allows the generation of stable gene modifications, which was not possible by using RNA silencing, the only previously available reverse genetic tool.

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Fig. 1: Schematic representation of the overall methodology for genetic manipulation of A. castellanii and GVs.
Fig. 2: Design of homology arms for gene modification by homologous recombination.
Fig. 3: Cloning of polycistronic DNA sequences for gRNA expression.
Fig. 4: Design of primers for genotyping.

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Data availability

All data supporting this paper are included within the article in the form of figures and tables. Raw data associated with Fig. 4 are provided in the primary articles15,18. All plasmids were deposited in Addgene (reference number indicated in Table 1).

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Acknowledgements

We thank Sandra Jeudy for helpful advice during the development of the protocol. This study was founded by the European Research Council under the European Union’s Horizon 2020 research and innovation program (grant agreement no. 832601; to C.A.). H.B. is the recipient of an EMBO Long-Term Fellowship (ALTF 979-2019).

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Author notes

  1. These authors contributed equally: Nadege Philippe, Hugo Bisio.

Authors and Affiliations

  1. Aix–Marseille University, Centre National de la Recherche Scientifique, Information Genomique & Structurale, Unite Mixte de Recherche 7256 (Institut de Microbiologie de la Mediterranee, FR3479, IM2B), Marseille, France

    Nadege Philippe, Avi Shukla, Chantal Abergel & Hugo Bisio

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  1. Nadege Philippe
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Contributions

H.B., N.P. and A.S. wrote the original draft. C.A. administered the project, acquired funding and wrote the original draft.

Corresponding authors

Correspondence to Chantal Abergel or Hugo Bisio.

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The authors declare no competing interests.

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Nature Protocols thanks Julien Andreani, Frank Aylward, Matthias Fischer, Hiroyuki Hikida, Abdeali Jivaji, Vasudha Sharma and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Related links

Key references using this protocol

Liu, Y. et al. Cell 184, 4237–4250.e19 (2021): https://doi.org/10.1016/j.cell.2021.06.032

Bisio, H. et al. Nat. Commun. 14, 428 (2023): https://doi.org/10.1038/s41467-023-36145-4

Alempic, J.-M. et al. Preprint at bioRxiv (2023): https://doi.org/10.1101/2023.04.28.538727

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Philippe, N., Shukla, A., Abergel, C. et al. Genetic manipulation of giant viruses and their host, Acanthamoeba castellanii. Nat Protoc 19, 3–29 (2024). https://doi.org/10.1038/s41596-023-00910-y

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