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Gene Editing in Chlamydomonas Using the SCREAM Technique
1Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia.
Bio-Protocol
|July 28, 2026
Summary
The SCREAM technique allows for repeated gene editing in Chlamydomonas reinhardtii using a single endogenous marker, simplifying complex genetic modifications. This method facilitates successive mutations for comprehensive gene function analysis.
Area of Science:
- * Molecular Biology
- * Genetics
- * Algal Biotechnology
Background:
- * CRISPR-Cas gene editing in *Chlamydomonas reinhardtii* allows for gene insertion, knockout, and mutation introduction.
- * Current methods rely on selection markers that are limited in number, require specific media, and are difficult to remove, complicating successive edits.
- * The low efficiency of gene editing necessitates marker enrichment before screening, adding complexity to multi-step modifications.
Purpose of the Study:
- * To develop a novel gene editing strategy in *Chlamydomonas reinhardtii* that overcomes the limitations of traditional selection markers.
- * To enable efficient and repeated gene editing in a single cell line using a recyclable endogenous marker.
- * To facilitate the creation of complex mutations for detailed gene function studies.
Main Methods:
- * Introduction of the SCREAM (sequential CRISPR via recycling endogenous auxotrophic markers) technique.
- * Utilization of an endogenous gene as a selectable marker, allowing for both loss-of-function and gain-of-function selection.
- * Sequential gene editing steps involving mutation of the marker and target genes, followed by marker reversion to wild-type status.
- * Employment of homology-directed repair with single-stranded oligonucleotides for precise genetic modifications.
Main Results:
- * Demonstrated the successful application of the SCREAM technique using nitrate reductase as the endogenous marker and adenine phosphoribosyltransferase as the target gene.
- * Showcased the ability to perform successive gene edits in a single cell line with indefinite reusability of the single endogenous marker.
- * Confirmed the creation of site-directed mutants, gene knockouts, and gene restoration, with only the target gene altered in the final strains.
Conclusions:
- * The SCREAM technique provides a versatile and efficient platform for complex genetic engineering in *Chlamydomonas reinhardtii*.
- * This method simplifies multi-step gene editing by enabling the reuse of a single endogenous auxotrophic marker, reducing the need for multiple selection media.
- * SCREAM is widely applicable to existing cell lines and facilitates functional analysis of genes, including redundant gene families.
