Affordable CRISPR RNP‐Based Genome Editing in <i>Euglena gracilis</i>

Genome editing can enhance basic research and enable industrial applications of green algae. Here, we present an affordable, broadly applicable workflow for genome editing in the unicellular green alga Euglena gracilis using Cas9 nucleases. This method retains high editing efficiency while significantly lowering technical barriers. Unlike previous approaches that required specialized equipment, this protocol can be performed using a general-purpose laboratory electroporator and a simplified clonal isolation procedure without the need for specialized micromanipulation devices. This protocol is compatible with a range of editing outcomes, such as targeted deletions and precise base substitutions, enabling more widespread genome editing in Euglena. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Culture of Euglena gracilis Basic Protocol 2: sgRNA synthesis Basic protocol 3: Transformation Basic protocol 4: Genotyping.