Targeted integration by homologous recombination enablesin situtagging and replacement of genes in the marine microeukaryoteDiplonema papillatum

0537342 - BC 2021 RIV GB eng J - Journal Article
Faktorová, Drahomíra - Kaur, Binnypreet - Valach, M. - Graf, L. - Benz, Corinna - Burger, G. - Lukeš, Julius
Targeted integration by homologous recombination enablesin situtagging and replacement of genes in the marine microeukaryoteDiplonema papillatum.
Environmental Microbiology. Roč. 22, č. 9 (2020), s. 3660-3670. ISSN 1462-2912. E-ISSN 1462-2920
R&D Projects: GA ČR(CZ) GA20-07186S; GA MŠMT(CZ) LL1601; GA MŠMT(CZ) EF16_019/0000759
Grant - others:Gordon and Betty Moore Foundation(US) GBMF4983.01
Program: Science
Institutional support: RVO:60077344
Keywords : dna * repair * diplonemids * expression * phylogeny * diversity * deletion * genomes * system * ku
OECD category: Biochemistry and molecular biology
Impact factor: 5.491, year: 2020
Method of publishing: Open access
https://sfamjournals.onlinelibrary.wiley.com/doi/10.1111/1462-2920.15130

Diplonemids are a group of highly diverse and abundant marine microeukaryotes that belong to the phylum Euglenozoa and form a sister clade to the well-studied, mostly parasitic kinetoplastids. Very little is known about the biology of diplonemids, as few species have been formally described and just one,Diplonema papillatum, has been studied to a decent extent at the molecular level. Following up on our previous results showing stable but random integration of delivered extraneous DNA, we demonstrate here homologous recombination inD. papillatum. Targeting various constructs to the intended position in the nuclear genome was successful when 5 ' and 3 ' homologous regions longer than 1 kbp were used, achieving N-terminal tagging with mCherry and gene replacement of alpha- and beta-tubulins. For more convenient genetic manipulation, we designed a modular plasmid, pDP002, which bears a protein-A tag and used it to generate and express a C-terminally tagged mitoribosomal protein. Lastly, we developed an improved transformation protocol for broader applicability across laboratories. Our robust methodology allows the replacement, integration as well as endogenous tagging ofD. papillatumgenes, thus opening the door to functional studies in this species and establishing a basic toolkit for reverse genetics of diplonemids in general.
Permanent Link: http://hdl.handle.net/11104/0315069