Unrestrained markerless trait stacking in Nannochloropsis gaditana through combined genome editing and marker recycling technologies
| Autor: | John H. Verruto, Melisa C. Low, Gregory Hannum, William F. Lambert, Jessica Nichole Greiner, Eric R. Moellering, Nicholas Bauman, Fedor I. Kuzminov, Jay McCarren, Sarah Tacke, Kristie Francis, Imad Ajjawi, Yingjun Wang, Ryan Kalb |
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| Rok vydání: | 2018 |
| Předmět: |
0106 biological sciences
0301 basic medicine Multidisciplinary Nuclear gene Transgene Cre recombinase Computational biology Biology 01 natural sciences 03 medical and health sciences 030104 developmental biology Genome editing Gene family Gene Gene knockout Selectable marker 010606 plant biology & botany |
| Zdroj: | Proceedings of the National Academy of Sciences. 115 |
| ISSN: | 1091-6490 0027-8424 |
| Popis: | Robust molecular tool kits in model and industrial microalgae are key to efficient targeted manipulation of endogenous and foreign genes in the nuclear genome for basic research and, as importantly, for the development of algal strains to produce renewable products such as biofuels. While Cas9-mediated gene knockout has been demonstrated in a small number of algal species with varying efficiency, the ability to stack traits or generate knockout mutations in two or more loci are often severely limited by selectable agent availability. This poses a critical hurdle in developing production strains, which require stacking of multiple traits, or in probing functionally redundant gene families. Here, we combine Cas9 genome editing with an inducible Cre recombinase in the industrial alga Nannochloropsis gaditana to generate a strain, NgCas9+Cre+, in which the potentially unlimited stacking of knockouts and addition of new genes is readily achievable. Cre-mediated marker recycling is first demonstrated in the removal of the selectable marker and GFP reporter transgenes associated with the Cas9/Cre construct in NgCas9+Cre+ Next, we show the proof-of-concept generation of a markerless knockout in a gene encoding an acyl-CoA oxidase (Aco1), as well as the markerless recapitulation of a 2-kb insert in the ZnCys gene 5'-UTR, which results in a doubling of wild-type lipid productivity. Finally, through an industrially oriented process, we generate mutants that exhibit up to ∼50% reduction in photosynthetic antennae size by markerless knockout of seven genes in the large light-harvesting complex gene family. |
| Databáze: | OpenAIRE |
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