Knockout 7 genes using Cas9 stacking traits

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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.

• Verruto J, Francis K, Wang Y, Low MC, Greiner J, Tacke S, Kuzminov F, Lambert W, McCarren J, Ajjawi I, Bauman N, Kalb R, Hannum G, Moellering ER. Unrestrained markerless trait stacking in. Proceedings of the National Academy of Sciences of the United States of America. 2018 07 24; 115(30):E7015-E7022.