CRISPR/Cas9 mutagenesis in Volvox carteri

Area of Science:

• Algal genetics
• Developmental biology
• Gene editing technologies

Background:

• Volvox carteri serves as a model organism for studying the evolution of developmental complexity.
• Previous genetic analysis in Volvox carteri has been limited by the lack of efficient targeted mutagenesis tools.

Purpose of the Study:

• To establish a CRISPR/Cas9-based system for targeted mutagenesis in Volvox carteri.
• To demonstrate the efficacy of this system for generating specific mutations in key developmental genes.

Main Methods:

• Conditional expression of Streptococcus pyogenes Cas9 and constitutive expression of single-guide RNA (sgRNA) using Volvox carteri regulatory sequences.
• Bombardment of Volvox carteri with Cas9 and sgRNA vectors targeting glsA, regA, and invA genes.
• Selection of transformants using a hygromycin-resistance marker and verification of mutations through phenotypic analysis and DNA sequencing.

Main Results:

• Successfully generated targeted frameshift mutations in glsA, regA, and invA genes in Volvox carteri.
• Observed expected mutant phenotypes, including gonidialess (Gls) phenotype for glsA mutants.
• Achieved mutation rates ranging from approximately 0.01% to 100% depending on the targeted gene and strain.

Conclusions:

• The developed CRISPR/Cas9 system provides a powerful and efficient method for targeted mutagenesis in Volvox carteri.
• This advancement will significantly accelerate molecular genetic studies of development and evolutionary novelty in volvocine algae.