Affordable CRISPR RNP-Based Genome Editing in Euglena gracilis

Area of Science:

• Algal biotechnology
• Molecular biology
• Synthetic biology

Background:

• Genome editing in green algae like Euglena gracilis is crucial for advancing basic research and industrial applications.
• Previous genome editing methods for Euglena gracilis were often technically demanding and required specialized equipment, limiting their widespread adoption.

Purpose of the Study:

• To present an affordable and broadly applicable workflow for genome editing in Euglena gracilis.
• To lower the technical barriers associated with genome editing in this organism.
• To enable a wider range of genome editing outcomes, including deletions and base substitutions.

Main Methods:

• Utilized Cas9 nucleases for genome editing in Euglena gracilis.
• Employed a general-purpose laboratory electroporator for transformation.
• Implemented a simplified clonal isolation procedure, avoiding specialized micromanipulation devices.
• Detailed protocols for Euglena gracilis culture, sgRNA synthesis, transformation, and genotyping were established.

Main Results:

• Achieved high genome editing efficiency in Euglena gracilis.
• Demonstrated the compatibility of the workflow with various editing outcomes, such as targeted deletions and precise base substitutions.
• Successfully adapted the protocol for use with standard laboratory equipment.

Conclusions:

• The presented workflow significantly lowers technical barriers for genome editing in Euglena gracilis.
• This accessible method facilitates broader application of genome editing for both fundamental research and industrial purposes in green algae.
• The protocol supports diverse editing strategies, paving the way for enhanced genetic manipulation of Euglena gracilis.