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Updated: Jun 20, 2026

High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii

High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii

Published on: December 5, 2016

Efficient methods for multiple types of precise gene-editing in Chlamydomonas.

Hui Chen¹, Qing-Lin Yang¹, Jia-Xi Xu^1,2

¹State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, 200031, China.

The Plant Journal : for Cell and Molecular Biology

|June 13, 2023

Summary

This study introduces precise CRISPR/Cas9 gene editing for Chlamydomonas reinhardtii, enabling gene inactivation, tagging, and precise modifications. These advancements enhance the utility of this model organism for research and industry.

Keywords:

Chlamydomonas efficient gene-editing homology-mediated multi-type precision

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Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Published on: May 25, 2018

Area of Science:

* Molecular Biology
* Genetics
* Algal Biotechnology

Background:

* Chlamydomonas reinhardtii is a model organism for photosynthesis and cilia research.
* Precise gene editing in Chlamydomonas is challenging, particularly for low-expression genes.
* Existing methods lack efficiency for complex genetic manipulations.

Purpose of the Study:

* To develop and validate a versatile CRISPR/Cas9-based gene editing system in Chlamydomonas reinhardtii.
* To demonstrate precise gene inactivation, epitope tagging, amino acid substitution, and fragment deletion.
* To enhance the genetic manipulation toolkit for Chlamydomonas research and applications.

Main Methods:

* Utilized CRISPR/Cas9 nuclease to create DNA breaks.
* Employed homologous DNA templates for precise repair-mediated editing.
* Applied the method for gene inactivation, FLAG-HA and YFP tagging, single amino acid substitution, and 3'-UTR fragment deletion.

Main Results:

* Successfully inactivated low-expression genes (CrTET1, CrKU80).
* Introduced epitope tags (FLAG-HA, YFP) into multiple genes (VIPP1, IFT46, CrTET1, CrKU80).
* Achieved precise single amino acid substitutions (FLA3, FLA10, FTSY) and stable gene knock-down via 3'-UTR deletion (MAA7, VIPP1).

Conclusions:

* Established efficient, multi-type precise gene editing methods for Chlamydomonas.
* Enabled base-resolution DNA modification (substitution, insertion, deletion).
* Expanded the potential of Chlamydomonas for fundamental research and industrial biotechnology.