Endogenous gene tagging in the model brown alga Ectocarpus using a simplified CRISPR/Cas method

Abstract

Brown algae represent one of the few eukaryotic lineages to have independently evolved complex multicellularity, providing a powerful comparative system for investigating the molecular and evolutionary principles underlying multicellular development. Ectocarpus has emerged as the principal model for this lineage, supported by extensive genomic and transcriptomic resources. However, mechanistic and functional studies have remained limited by the available reverse-genetic tools. While recent CRISPR-Cas developments have enabled targeted gene knock-outs, the lack of knock-in (KI) approaches for endogenous protein tagging and precise genomic insertion remains a major experimental bottleneck. Here, we establish a comprehensive CRISPR-Cas genome-engineering framework for Ectocarpus that enables both targeted gene disruption and precise genomic insertion. We demonstrate efficient knock-in of multiple peptide tags at endogenous loci, enabling direct analysis of native proteins. By combining robust gene knock-out with endogenous protein tagging, this framework substantially expands the experimental possibilities for brown algal research and establishes Ectocarpus as a genetically tractable system for functional genomics, providing a foundation for genome engineering across stramenopiles.