The Challenge of Limited Tools for Microalgae Mutation

Microalgae have untapped potential but are hindered by a lack of effective tools to create loss-of-function mutants. Additionally, for field deployment, modified strains must be free of antibiotic resistance genes and face fewer regulatory hurdles as transgene-free organisms.

Nannochloropsis oceanica CCMP1779: A Model for Lipid Metabolism

Nannochloropsis oceanica CCMP1779, an oleaginous microalga, is emerging as a valuable model for studying microalgal lipid metabolism. Its unique properties make it an ideal candidate for advancements in genetic engineering.

Introducing a One-Vector Episomal CRISPR/Cas9 System

We introduce a cutting-edge one-vector episomal CRISPR/Cas9 system designed specifically for Nannochloropsis oceanica. This innovative system allows for the generation of marker-free mutant lines.

The Power of the CRISPR/Cas9 System

The vector used in this system includes the CEN/ARS6 region from Saccharomyces cerevisiae, ensuring the maintenance of the circular extrachromosomal DNA. With a bidirectional promoter, the vector produces both Cas9 and a ribozyme-flanked sgRNA. This powerful combination efficiently generates targeted mutations.

Marker-Free Nontransgenic Engineered Lines

One significant advantage of our system is the ability to remove the episomal DNA after selection pressure is eliminated, resulting in marker-free nontransgenic engineered lines. This feature ensures the absence of unwanted genetic material, allowing for safer and more efficient applications.

Demonstrating the System: Nitrate Reductase Gene Disruption

To validate the effectiveness of this system, we successfully disrupted the nitrate reductase gene (NR) in Nannochloropsis oceanica CCMP1779. Subsequently, we removed the CRISPR episome, resulting in the generation of nontransgenic marker-free nitrate reductase knockout lines (NR-KO).