Comprehensive Genome Engineering Toolbox for Microalgae Nannochloropsis oceanica Based on CRISPR-Cas Systems

Area of Science:

• Synthetic biology
• Molecular biology
• Biotechnology

Background:

• Microalgae are valuable for producing metabolites using CO2 and sunlight.
• Advanced molecular tools are needed for high-throughput genome engineering in microalgae.
• Current methods for microalgal strain improvement are limited.

Purpose of the Study:

• To develop novel genome editing tools for microalgae.
• To enable scarless and markerless gene editing in *Nannochloropsis oceanica*.
• To establish multiplexed genome engineering and gene downregulation capabilities.

Main Methods:

• Utilized Cas12a ribonucleoproteins (RNPs) with homology-directed repair (HDR) for precise editing.
• Developed an episomal plasmid-based Cas12a system for efficient indel introduction.
• Implemented a CRISPR interference (CRISPRi) system using dCas9 and Cas12a for gene silencing.
• Applied multiplexed genome engineering by targeting three genomic sites simultaneously.

Main Results:

• Achieved scarless and markerless genome editing in *N. oceanica*.
• Successfully introduced indels using an episomal Cas12a system.
• Demonstrated multiplexed genome engineering, targeting three sites in one transformation.
• Achieved significant gene downregulation (up to 85% transcript reduction) using CRISPRi.

Conclusions:

• Developed a robust toolbox for accelerating microalgal genome engineering.
• The new tools facilitate high-throughput strain development for improved industrial traits.
• These advancements hold potential for enhancing various microalgal species.