Cazier Andrew P, Son Jaewoo, Yellayi Sreenivas, Chavez Lizmarie S, Young Caden, Irvin Olivia M, Abraham Hannah, Dalvi Saachi, Blazeck John
School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA, USA.
Parker H. Petit Institute of Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA, USA.
Nat Commun. 2025 Jul 1;16(1):5688. doi: 10.1038/s41467-025-61206-1.
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.
V(D)J重排是抗体多样性产生所必需的过程，它通过由几种蛋白质介导的复杂DNA切割和修复过程进行，这些蛋白质包括重组激活基因1和2（RAG1和RAG2）。V(D)J重排发生在所有有颌脊椎动物中，但在进化关系较远的物种中不存在，包括酿酒酵母。由于酵母生长迅速且是抗体展示的平台，对酵母进行工程改造使其能够进行V(D)J重排，可以扩大其在抗体发育研究中的应用范围。因此，在这项工作中，我们将RAG1和RAG2引入酵母，并使用分裂抗生素抗性测定法来表征由此产生的重排能力，证明了编码接头的同源性辅助形成成功。通过采用多种策略，我们将同源性辅助重排的速率提高了7000多倍，四天后最佳速率接近1%的重排率。我们进一步表明，我们的平台可以检测几种致病RAG1突变的严重程度。最后，我们使用我们改造后的酵母，从一系列无功能的基因片段开始，同时生成多达三种独特的荧光蛋白或两种不同的抗体片段，我们认为这是首次仅利用非脊椎动物细胞中现有DNA的随机重组产生遗传和表型多样性。
