High-efficiency genome editing by Cas12a ribonucleoprotein complex in Euglena gracilis

Area of Science:

• Microalgal biotechnology
• Genome editing technologies
• Synthetic biology

Background:

• Euglena gracilis is a valuable microalga for food, feed, cosmetics, and biofuels.
• Limited genetic manipulation methods hinder Euglena gracilis research and industrial use.
• CRISPR-based genome editing offers potential for microalgal genetic engineering.

Purpose of the Study:

• To develop a high-efficiency, transgene-free genome editing method for Euglena gracilis.
• To complement existing Cas9 RNP-based methods with a novel LbCas12a RNP system.
• To expand genetic engineering capabilities in Euglena for research and bioproduction.

Main Methods:

• Utilized Lachnospiraceae bacterium CRISPR-associated protein 12a (LbCas12a) ribonucleoprotein (RNP) complexes.
• Direct delivery of LbCas12a-containing RNPs into Euglena gracilis.
• Employed single-stranded DNA donor templates for knock-in and base editing.

Main Results:

• Achieved high mutagenesis rates (77.2-94.5%) at EgGSL2 and EgcrtB genes in Euglena gracilis.
• Demonstrated efficient transgene-free genome editing using LbCas12a RNPs.
• Successfully performed targeted mutagenesis, knock-in, and base editing in Euglena gracilis.

Conclusions:

• The developed LbCas12a RNP method provides an efficient and versatile tool for Euglena gracilis genetic engineering.
• This advancement facilitates basic research and accelerates the engineering of Euglena for bioproduction.
• Expands the toolkit for microalgal genetic manipulation, supporting diverse applications.