Manipulating the 3D organization of the largest synthetic yeast chromosome
Weimin Zhang, Luciana Lazar‐Stefanita, Hitoyoshi Yamashita, Michael Shen, Leslie A. Mitchell, Hikaru Kurasawa, Evgenii Lobzaev, Viola Fanfani, Max A. B. Haase, Xiaoji Sun, Qingwen Jiang, Gregory W. Goldberg, David M. Ichikawa, Stephanie Lauer, Laura H. McCulloch, Nicole Easo, S. Jiaming Lin, Brendan Camellato, Yinan Zhu, Jitong Cai, Zhuwei Xu, Yu Zhao, Maya Sacasa, Ryan Accardo, Leighanne A. Brammer Basta, Nicholas R. Bello, Lousanna Cai, Stephanie Cerritos, MacIntosh Cornwell, Anthony D’Amato, Maria Hacker, Kenneth Hersey, Emma Kennedy, Ardeshir Kianercy, Do‐Hee Kim, Griffin McCutcheon, Kimiko McGirr, Nora Meaney, Maisa Nimer, Carla Sabbatini, Lisa Z. Scheifele, Lucas Shores, Cassandra Silvestrone, Arden Snee, Antonio Spina, Anthony Staiti, Matt Stuver, Elli Tian, Danielle Whearty, Calvin Zhao, Karen Zeller, Marcus B. Noyes, Joel S. Bader, Samuel Deutsch, Giovanni Stracquadanio, Yasunori Aizawa, Junbiao Dai, Jef D. Boeke
Abstract
Whether synthetic genomes can power life has attracted broad interest in the synthetic biology field. Here, we report de novo synthesis of the largest eukaryotic chromosome thus far, synIV, a 1,454,621-bp yeast chromosome resulting from extensive genome streamlining and modification. We developed megachunk assembly combined with a hierarchical integration strategy, which significantly increased the accuracy and flexibility of synthetic chromosome construction. Besides the drastic sequence changes, we further manipulated the 3D structure of synIV to explore spatial gene regulation. Surprisingly, we found few gene expression changes, suggesting that positioning inside the yeast nucleoplasm plays a minor role in gene regulation. Lastly, we tethered synIV to the inner nuclear membrane via its hundreds of loxPsym sites and observed transcriptional repression of the entire chromosome, demonstrating chromosome-wide transcription manipulation without changing the DNA sequences. Our manipulation of the spatial structure of synIV sheds light on higher-order architectural design of the synthetic genomes.