Generating combinatorial diversity via engineered V(D)J-like recombination in Saccharomyces cerevisiae.

Area of Science:

• Molecular Biology
• Immunology
• Synthetic Biology

Background:

• V(D)J recombination is essential for antibody diversity in jawed vertebrates.
• This process involves recombination-activating genes 1 and 2 (RAG1 and RAG2).
• Yeast, like Saccharomyces cerevisiae, lack V(D)J recombination but offer advantages for antibody display.

Purpose of the Study:

• To engineer yeast to undergo V(D)J recombination.
• To enhance the efficiency of homology-assisted recombination in yeast.
• To establish a platform for studying RAG1 mutations and generating genetic diversity.

Main Methods:

• Incorporation of RAG1 and RAG2 into Saccharomyces cerevisiae.
• Utilizing a split antibiotic resistance assay to measure recombination.
• Employing various strategies to optimize recombination rates.

Main Results:

• Demonstrated successful homology-assisted coding joint formation in engineered yeast.
• Achieved a 7000-fold increase in recombination rate, reaching nearly 1% after four days.
• Developed a platform to assess RAG1 mutations and generate diverse proteins and antibody fragments.

Conclusions:

• Engineered yeast can perform V(D)J recombination, expanding its utility beyond vertebrates.
• The developed platform facilitates the study of recombination mechanisms and disease-causing mutations.
• This work represents the first generation of genetic and phenotypic diversity via random recombination in a non-vertebrate cell.