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Updated: Feb 4, 2026

Genome Engineering of Primary Human B Cells Using CRISPR/Cas9
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Engineering Wax Ester Composition in Euglena gracilis Using Genome Editing.

Sakura Nagamine1, Rikuto Oishi1, Masami Nakazawa2

  • 1Department of Applied Biochemistry, Faculty of Agriculture, Osaka Metropolitan University, Sakai, Osaka, Japan.

Methods in Molecular Biology (Clifton, N.J.)
|February 2, 2026
PubMed
Summary
This summary is machine-generated.

This study details a CRISPR/Cas9 genome editing protocol for Euglena gracilis to modify wax ester production. This method enables stable genetic engineering of E. gracilis for synthetic biology and biomanufacturing applications.

Keywords:
Anaerobic metabolismCRISPR/Cas9Euglena gracilisFatty acid β-oxidationGenome editingLoss-of-functionMetabolic modificationTargeted gene knockoutWax ester synthesis

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Area of Science:

  • Synthetic Biology
  • Metabolic Engineering
  • Genomics

Background:

  • Genome editing advances enable metabolic engineering in non-model organisms like Euglena gracilis.
  • CRISPR/Cas9 and CRISPR/Cas12a systems have been established for E. gracilis genome editing.

Purpose of the Study:

  • To provide a detailed protocol for CRISPR/Cas9-based genome editing in E. gracilis.
  • To enable stable modification of wax ester composition under anaerobic conditions.

Main Methods:

  • Utilizing CRISPR/Cas9 technology to target key enzymes in the reversed β-oxidation pathway.
  • Generating knockout mutants to alter wax ester chain lengths in E. gracilis.

Main Results:

  • Successfully generated E. gracilis knockout mutants with altered wax ester chain lengths.
  • Established a reproducible and stable method for genetic modification of E. gracilis metabolism.

Conclusions:

  • The developed CRISPR/Cas9 protocol facilitates stable genetic modification of E. gracilis metabolism.
  • This approach supports the use of E. gracilis as a green chassis for synthetic biology and biomanufacturing, with potential for knock-in strategies.