TIM, a targeted insertional mutagenesis method utilizing CRISPR/Cas9 in Chlamydomonas reinhardtii

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Understanding gene and protein functions relies on mutant generation and analysis.
• CRISPR-based methods offer powerful tools for genetic manipulation.
• Efficient mutagenesis is crucial for model organisms like Chlamydomonas reinhardtii.

Purpose of the Study:

• To introduce TIM, a novel CRISPR-based targeted insertional mutagenesis method.
• To establish an efficient, cost-effective tool for Chlamydomonas reinhardtii research.
• To enhance the study of gene functions through improved mutagenesis.

Main Methods:

• Utilized CRISPR-Cas9 technology with guide RNA (gRNA) ribonucleoprotein (RNP) complexes.
• Delivered RNP and exogenous double-stranded DNA donor with homology arms and antibiotic resistance gene.
• Optimized parameters for delivery via electroporation or glass-bead method.

Main Results:

• Achieved mutation efficiencies of 40% to 95% for six different genes in two strains.
• Successfully generated simultaneous mutations in two separate genes in a single experiment.
• Demonstrated high mutation rates superior to previous CRISPR-based methods in C. reinhardtii.

Conclusions:

• TIM is an efficient and versatile CRISPR-based method for targeted insertional mutagenesis in Chlamydomonas reinhardtii.
• The method offers high mutation rates and flexibility, facilitating gene function studies.
• TIM is expected to be effective for targeting numerous non-essential nuclear genes.