Summary
Whether synthetic genomes can power life has attracted broad interest in the synthetic biology field, especially when the synthetic genomes are extensively modified with thousands of designer features. Here we report de novo synthesis of the largest eukaryotic chromosome thus far, synIV, a 1,454,621-bp Saccharomyces cerevisiae chromosome resulting from extensive genome streamlining and modification. During the construction of synIV, we developed megachunk assembly combined with a hierarchical integration strategy, which significantly increased the accuracy and flexibility of synthetic chromosome construction and facilitated chromosome debugging. In addition to the drastic sequence changes made to synIV by rewriting it, we further manipulated the three-dimensional structure of synIV in the yeast nucleus to explore spatial gene regulation within the nuclear space. 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 the largest synthetic yeast chromosome shed light on higher-order architectural design of the synthetic genomes.
Highlights
De novo synthesis of the largest eukaryotic chromosome, synIV
SynIV shows similar 3D structure to wild-type IV, despite thousands of changes made to it
“Inside-out” repositioning of synIV in nucleus shows minor transcriptional changes
Multipoint tethering synIV to inner nuclear membrane represses transcription of whole chromosome
Competing Interest Statement
Jef Boeke is a Founder and Director of CDI Labs, Inc., a Founder of and consultant to Neochromosome, Inc, a Founder, SAB member of and consultant to ReOpen Diagnostics, LLC and serves or served on the Scientific Advisory Board of the following: Sangamo, Inc., Modern Meadow, Inc., Rome Therapeutics, Inc., Sample6, Inc., Tessera Therapeutics, Inc. and the Wyss Institute. Yasunori Aizawa is a Founder and CSO of Logomix, Inc.
Footnotes
↵# Build a Genome class
New results are added to the revised manuscript demonstrating whole chromosome tethering to the inner nuclear membrane, thus, selectively silencing the entire or partial synthetic chromosome. We showed the feasibility of regulating genome expression by manipulating the 3D organization of it and leaving the DNA sequence untouched.
