Docampo Roberto

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Docampo
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Roberto
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  • Article
    CRISPR/Cas9-induced disruption of Bodo saltans paraflagellar rod-2 gene reveals its importance for cell survival
    (Society for Applied Microbiology, 2022-01-31) Gomaa, Fatma ; Li, Zhu-Hong ; Beaudoin, David J. ; Alzan, Heba ; Girguis, Peter R. ; Docampo, Roberto ; Edgcomb, Virginia P.
    Developing transfection protocols for marine protists is an emerging field that will allow the functional characterization of protist genes and their roles in organism responses to the environment. We developed a CRISPR/Cas9 editing protocol for Bodo saltans, a free-living kinetoplastid with tolerance to both marine and freshwater conditions and a close non-parasitic relative of trypanosomatids. Our results show that SaCas9/single-guide RNA (sgRNA) ribonucleoprotein (RNP) complex-mediated disruption of the paraflagellar rod 2 gene (BsPFR2) was achieved using electroporation-mediated transfection. The use of CRISPR/Cas9 genome editing can increase the efficiency of targeted homologous recombination when a repair DNA template is provided. Our sequence analysis suggests two mechanisms for repairing double-strand breaks in B. saltans are active; homologous-directed repair (HDR) utilizing an exogenous DNA template that carries an antibiotic resistance gene and likley non-homologous end joining (NHEJ). However, HDR was only achieved when a single (vs. multiple) SaCas9 RNP complex was provided. Furthermore, the biallelic knockout of BsPFR2 was detrimental for the cell, highlighting its essential role for cell survival because it facilitates the movement of food particles into the cytostome. Our Cas9/sgRNA RNP complex protocol provides a new tool for assessing gene functions in B. saltans and perhaps similar protists with polycistronic transcription.
  • Article
    Genetic tool development in marine protists: emerging model organisms for experimental cell biology
    (Nature Research, 2020-04-06) Faktorová, Drahomíra ; Nisbet, R. Ellen R. ; Fernández Robledo, José A. ; Casacuberta, Elena ; Sudek, Lisa ; Allen, Andrew E. ; Ares, Manuel, Jr. ; Aresté, Cristina ; Balestreri, Cecilia ; Barbrook, Adrian C. ; Beardslee, Patrick ; Bender, Sara J. ; Booth, David S. ; Bouget, François-Yves ; Bowler, Chris ; Breglia, Susana A. ; Brownlee, Colin ; Burger, Gertraud ; Cerutti, Heriberto ; Cesaroni, Rachele ; Chiurillo, Miguel A. ; Clemente, Thomas ; Coles, Duncan B. ; Collier, Jackie L. ; Cooney, Elizabeth C. ; Coyne, Kathryn J. ; Docampo, Roberto ; Dupont, Christopher L. ; Edgcomb, Virginia P. ; Einarsso, Elin ; Elustondo, Pía A. ; Federici, Fernan ; Freire-Beneitez, Veronica ; Freyria, Nastasia J. ; Fukuda, Kodai ; García, Paulo A. ; Girguis, Peter R. ; Gomaa, Fatma ; Gornik, Sebastian G. ; Guo, Jian ; Hampl, Vladimír ; Hanawa, Yutaka ; Haro-Contreras, Esteban R. ; Hehenberger, Elisabeth ; Highfield, Andrea ; Hirakawa, Yoshihisa ; Hopes, Amanda ; Howe, Christopher J. ; Hu, Ian ; Ibañez, Jorge ; Irwin, Nicholas A. T. ; Ishii, Yuu ; Janowicz, Natalia Ewa ; Jones, Adam C. ; Kachale, Ambar ; Fujimura-Kamada, Konomi ; Kaur, Binnypreet ; Kaye, Jonathan Z. ; Kazana, Eleanna ; Keeling, Patrick J. ; King, Nicole ; Klobutcher, Lawrence A. ; Lander, Noelia ; Lassadi, Imen ; Li, Zhuhong ; Lin, Senjie ; Lozano, Jean-Claude ; Luan, Fulei ; Maruyama, Shinichiro ; Matute, Tamara ; Miceli, Cristina ; Minagawa, Jun ; Moosburner, Mark ; Najle, Sebastián R. ; Nanjappa, Deepak ; Nimmo, Isabel C. ; Noble, Luke ; Novák Vanclová, Anna M. G. ; Nowacki, Mariusz ; Nuñez, Isaac ; Pain, Arnab ; Piersanti, Angela ; Pucciarelli, Sandra ; Pyrih, Jan ; Rest, Joshua S. ; Rius, Mariana ; Robertson, Deborah ; Ruaud, Albane ; Ruiz-Trillo, Iñaki ; Sigg, Monika A. ; Silver, Pamela A. ; Slamovits, Claudio H. ; Smith, G. Jason ; Sprecher, Brittany N. ; Stern, Rowena ; Swart, Estienne C. ; Tsaousis, Anastasios D. ; Tsypin, Lev ; Turkewitz, Aaron ; Turnšek, Jernej ; Valach, Matus ; Vergé, Valérie ; von Dassow, Peter ; von der Haar, Tobias ; Waller, Ross F. ; Wang, Lu ; Wen, Xiaoxue ; Wheeler, Glen L. ; Woods, April ; Zhang, Huan ; Mock, Thomas ; Worden, Alexandra Z. ; Lukes, Julius
    Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways.