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

TL;DR

Researchers engineered yeast to perform V(D)J recombination, enabling the creation of antibody diversity and combinatorial genetic variation in a non-vertebrate organism.

Contribution

The first demonstration of V(D)J recombination in yeast, enabling combinatorial diversity generation from preexisting DNA fragments.

Findings

Yeast expressing RAG1 and RAG2 can form coding joints through homology-assisted recombination.

Recombination rates were increased over 7000-fold, reaching up to 1% after four days.

The system can generate multiple unique proteins or antibody fragments from nonfunctional gene fragments.

Abstract

V(D)J recombination is integral to the development of antibody diversity and proceeds through a complex DNA cleavage and repair process mediated by several proteins, including recombination-activating genes 1 and 2, RAG1 and RAG2. V(D)J recombination occurs in all jawed vertebrates but is absent from evolutionarily distant relatives, including the yeast Saccharomyces cerevisiae. As yeast grow quickly and are a platform for antibody display, engineering yeast to undergo V(D)J recombination could expand their applicability for studying antibody development. Therefore, in this work we incorporate RAG1 and RAG2 into yeast and characterize the resulting recombination ability using a split antibiotic resistance assay, demonstrating successful homology-assisted formation of coding joints. By pursuing a variety of strategies, we increase the rate of homology-assisted recombination by over 7000-fold, with the best rates approaching 1% recombination after four days. We further show that our platform can assay the severity of several disease-causing RAG1 mutations. Finally, we use our engineered yeast to simultaneously generate up to three unique fluorescent proteins or two distinct antibody fragments starting from an array of nonfunctional gene fragments, which we believe to be the first-ever generation of genetic and phenotypic diversity solely using random recombination of preexisting DNA in a non-vertebrate cell.

Mammalian recombination activating genes RAG1 and RAG2 are essential for the production and diversification of antibodies and T-cell receptors via V(D)J recombination in lymphocytes but are absent in simpler eukaryotes such as the yeast Saccharomyces cerevisiae. Here the authors integrate mouse RAG1/2 in S. cerevisiae and demonstrate the ability to create combinatorial diversity starting from a single genetic locus in vivo.

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