Manipulating the 3D organization of the largest synthetic yeast chromosome

Abstract

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 a megachunk assembly method, combined with a hierarchical integration strategy. This strategy 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. Therefore, our manipulation of the spatial structure of the largest synthetic yeast chromosome shed light on higher-order architectural design of the synthetic genomes.

Synthetic genome modules designed for programmable silencing of functions and chromosomes

“…However, we found this method to be less efficient than dCre-SIR and also caused fitness defects. A recent study employed a similar strategy but, in this case, attempted to tether the entire synthetic yeast chromosome synIV to the inner nuclear membrane (INM) using a custom ZF-Heh1-Heh2 fusion protein designed to target the loxPsym sequences 37 . Their chromosomal tethering approach showed significant growth defects upon induction, possibly due to ~2-fold downregulation of chromosomal genes, particularly essential genes 37 .…”

“…A recent study employed a similar strategy but, in this case, attempted to tether the entire synthetic yeast chromosome synIV to the inner nuclear membrane (INM) using a custom ZF-Heh1-Heh2 fusion protein designed to target the loxPsym sequences 37 . Their chromosomal tethering approach showed significant growth defects upon induction, possibly due to ~2-fold downregulation of chromosomal genes, particularly essential genes 37 . Consistent with our findings, a modest growth defect was observed when expressing either ZF-Heh1 or ZF-Heh2 alone, indicating potential off-target effects 37 .…”

“…Their chromosomal tethering approach showed significant growth defects upon induction, possibly due to ∼2-fold downregulation of chromosomal genes, particularly essential genes 37 . Consistent with our findings, a modest growth defect was observed when expressing either ZF-Heh1 or ZF-Heh2 alone, indicating potential off-target effects 37 . These off-targeting effects could be attributed to the non-specific binding of DBD or the correlation of Heh1/Heh2 with other essential cellular functions.…”

“…To enable external control of the functions of synthetic genome modules, we next sought to develop a synthetic system that can give 'master switch' regulation over all genes in a module, without needing to alter the DNA sequence of the genes and their promoters. For this we turned to the long-range regulation afforded by epigenetic control and attempted two main strategies: (1) the directed recruitment of chromatin regulators to the synthetic module DNA to initiate and maintain stable chromatin states 27 ; and, (2) the strategic tethering of module DNA into sub-nuclear regions associated with heterochromatin, such as the nuclear periphery, in order to leverage spatial gene regulation [35][36][37] .…”

“…An alternative strategy for synthetically modulating regulatory states of the synthetic modules is to physically tether them to native silencing foci. Programmable regulation by spatial redesign of the genome has been demonstrated in yeast cells and mammalian cells using CRISPR-Cas technologies and other DNA-protein binding systems [35][36][37][48][49][50][51] . However, the functional roles of these tethering approaches have led to mixed results.…”