Design, construction, and functional characterization of a tRNA neochromosome in yeast.

Here, we report the design, construction, and characterization of a tRNA neochromosome, a designer chromosome that functions as an additional, de novo counterpart to the native complement of Saccharomyces cerevisiae. Intending to address one of the central design principles of the Sc2.0 project, the ∼190-kb tRNA neochromosome houses all 275 relocated nuclear tRNA genes. To maximize stability, the design incorporates orthogonal genetic elements from non-S. cerevisiae yeast species. Furthermore, the presence of 283 rox recombination sites enables an orthogonal tRNA SCRaMbLE system. Following construction in yeast, we obtained evidence of a potent selective force, manifesting as a spontaneous doubling in cell ploidy. Furthermore, tRNA sequencing, transcriptomics, proteomics, nucleosome mapping, replication profiling, FISH, and Hi-C were undertaken to investigate questions of tRNA neochromosome behavior and function. Its construction demonstrates the remarkable tractability of the yeast model and opens up opportunities to directly test hypotheses surrounding these essential non-coding RNAs.

Engineering and functional analysis of yeast with a monotypic 40S ribosome subunit.

Emerging evidence reveals that ribosomes are not monolithic but dynamic machines with heterogeneous protein compositions that can reshape ribosomal translational abilities and cellular adaptation to environmental changes. Duplications of ribosomal protein (RP) genes are ubiquitous among organisms and are believed to affect cell function through paralog-specific regulation (e.g., by generating heterogeneous ribosomes) and/or gene dose amplification. However, direct evaluations of their impacts on cell function remain elusive due to the highly heterogeneous cellular RP pool. Here, we engineered a yeast with homogeneous 40S RP paralog compositions, designated homo-40S, by deleting the entire set of alternative duplicated genes encoding yeast 40S RP paralogs. Homo-40S displayed mild growth defects along with high sensitivity to the translation inhibitor paromomycin and a significantly increased stop codon readthrough. Moreover, doubling of the remaining RP paralogous genes in homo-40S rescued these phenotypes markedly, although not fully, compared to the wild-type phenotype, indicating that the dose of 40S RP genes together with the heterogeneity of the contents was vital for maintaining normal translational functionalities and growth robustness. Additional experiments revealed that homo-40S improved paromomycin tolerance via acquisition of bypass mutations or evolved to be diploid to generate fast-growing derivatives, highlighting the mutational robustness of engineered yeast to accommodate environmental and genetic changes. In summary, our work demonstrated that duplicated RP paralogs impart robustness and phenotypic plasticity through both gene dose amplification and paralog-specific regulation, paving the way for the direct study of ribosome biology through monotypic ribosomes with a homogeneous composition of specific RP paralogs.

LncRNA-Smad7 mediates cross-talk between Nodal/TGF-ß and BMP signaling to regulate cell fate determination of pluripotent and multipotent cells.

Transforming growth factor ß (TGF-ß) superfamily proteins are potent regulators of cellular development and differentiation. Nodal/Activin/TGF-ß and BMP ligands are both present in the intra- and extracellular milieu during early development, and cross-talk between these two branches of developmental signaling is currently the subject of intense research focus. Here, we show that the Nodal induced lncRNA-Smad7 regulates cell fate determination via repression of BMP signaling in mouse embryonic stem cells (mESCs). Depletion of lncRNA-Smad7 dramatically impairs cardiomyocyte differentiation in mESCs. Moreover, lncRNA-Smad7 represses Bmp2 expression through binding with the Bmp2 promoter region via (CA)12-repeats that forms an R-loop. Importantly, Bmp2 knockdown rescues defects in cardiomyocyte differentiation induced by lncRNA-Smad7 knockdown. Hence, lncRNA-Smad7 antagonizes BMP signaling in mESCs, and similarly regulates cell fate determination between osteocyte and myocyte formation in C2C12 mouse myoblasts. Moreover, lncRNA-Smad7 associates with hnRNPK in mESCs and hnRNPK binds at the Bmp2 promoter, potentially contributing to Bmp2 expression repression. The antagonistic effects between Nodal/TGF-ß and BMP signaling via lncRNA-Smad7 described in this work provides a framework for understanding cell fate determination in early development.

Large-scale pathway reconstruction and colorimetric screening accelerate cellular metabolism engineering.

The capability to manipulate and analyze hard-wired metabolic pathways sets the pace at which we can engineer cellular metabolism. Here, we present a framework to extensively rewrite the central metabolic pathway for malonyl-CoA biosynthesis in yeast and readily assess malonyl-CoA output based on pathway-scale DNA reconstruction in combination with colorimetric screening (Pracs). We applied Pracs to generate and test millions of enzyme variants by introducing genetic mutations into the whole set of genes encoding the malonyl-CoA biosynthetic pathway and identified hundreds of beneficial enzyme mutants with increased malonyl-CoA output. Furthermore, the synthetic pathways reconstructed by randomly integrating these beneficial enzyme variants generated vast phenotypic diversity, with some displaying higher production of malonyl-CoA as well as other metabolites, such as carotenoids and betaxanthin, thus demonstrating the generic utility of Pracs to efficiently orchestrate central metabolism to optimize the production of different chemicals in various metabolic pathways. Pracs will be broadly useful to advance our ability to understand and engineer cellular metabolism.

Probing eukaryotic genome functions with synthetic chromosomes.

The ability to redesign and reconstruct a cell at whole-genome level provides new platforms for biological study. The international synthetic yeast genome project-Sc2.0, designed by interrogating knowledge amassed by the yeast community to date, exemplifies how a classical synthetic biology "design-build-test-learn" engineering cycle can effectively test hypotheses about various genome fundamentals. The genome reshuffling SCRaMbLE system implemented in synthetic yeast strains also provides unprecedented diversified resources for genotype-phenotype study and yeast metabolic engineering. Further development of genome synthesis technology will shed new lights on complex biological processes in higher eukaryotes.

Cell Lysate Microarray for Mapping the Network of Genetic Regulators for Histone Marks.

Proteins, as the major executer for cell progresses and functions, its abundance and the level of post-translational modifications, are tightly monitored by regulators. Genetic perturbation could help us to understand the relationships between genes and protein functions. Herein, to explore the impact of the genome-wide interruption on certain protein, we developed a cell lysate microarray on kilo-conditions (CLICK) with 4837 knockout (YKO) and 322 temperature-sensitive (ts) mutant strains of yeast (Saccharomyces cerevisiae). Taking histone marks as examples, a general workflow was established for the global identification of upstream regulators. Through a single CLICK array test, we obtained a series of regulators for H3K4me3, which covers most of the known regulators in S. cerevisiae We also noted that several group of proteins are involved in negatively regulation of H3K4me3. Further, we discovered that Cab4p and Cab5p, two key enzymes of CoA biosynthesis, play central roles in histone acylation. Because of its general applicability, CLICK array could be easily adopted to rapid and global identification of upstream protein/enzyme(s) that regulate/modify the level of a protein or the posttranslational modification of a non-histone protein.

Multiplex Genome Engineering for Optimizing Bioproduction in Saccharomyces cerevisiae.

The field of synthetic biology is already beginning to realize its potential, with a wealth of examples showcasing the successful genetic engineering of microorganisms for the production of valuable compounds. The chassis Saccharomyces cerevisiae has been engineered to function as a microfactory for producing many of these economically and medically relevant compounds. However, strain construction and optimization to produce industrially relevant titers necessitate a wealth of underpinning biological knowledge alongside large investments of capital and time. Over the past decade, advances in DNA synthesis and editing tools have enabled multiplex genome engineering of yeast, permitting access to more complex modifications that could not have been easily generated in the past. These genome engineering efforts often result in large populations of strains with genetic diversity that can pose a significant challenge to screen individually via traditional methods such as mass spectrometry. The large number of samples generated would necessitate screening methods capable of analyzing all of the strains generated to maximize the explored genetic space. In this Perspective, we focus on recent innovations in multiplex genome engineering of S. cerevisiae, together with biosensors and high-throughput screening tools, such as droplet microfluidics, and their applications in accelerating chassis optimization.

Large-scale genomic rearrangements boost SCRaMbLE in Saccharomyces cerevisiae.

Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) is a promising tool to study genomic rearrangements. However, the potential of SCRaMbLE to study genomic rearrangements is currently hindered, because a strain containing all 16 synthetic chromosomes is not yet available. Here, we construct SparLox83R, a yeast strain containing 83 loxPsym sites distributed across all 16 chromosomes. SCRaMbLE of SparLox83R produces versatile genome-wide genomic rearrangements, including inter-chromosomal events. Moreover, when combined with synthetic chromosomes, SCRaMbLE of hetero-diploids with SparLox83R leads to increased diversity of genomic rearrangements and relatively faster evolution of traits compared to hetero-diploids only with wild-type chromosomes. Analysis of the SCRaMbLEd strain with increased tolerance to nocodazole demonstrates that genomic rearrangements can perturb the transcriptome and 3D genome structure and consequently impact phenotypes. In summary, a genome with sparsely distributed loxPsym sites can serve as a powerful tool for studying the consequence of genomic rearrangements and accelerating strain engineering in Saccharomyces cerevisiae.

High plasticity of ribosomal DNA organization in budding yeast.

In eukaryotic genomes, rDNA generally resides as a highly repetitive and dynamic structure, making it difficult to study. Here, a synthetic rDNA array on chromosome III in budding yeast was constructed to serve as the sole source of rRNA. Utilizing the loxPsym site within each rDNA repeat and the Cre recombinase, we were able to reduce the copy number to as few as eight copies. Additionally, we constructed strains with two or three rDNA arrays and found that the presence of multiple arrays did not affect the formation of a single nucleolus. Although alteration of the position and number of rDNA arrays did impact the three-dimensional genome structure, the additional rDNA arrays had no deleterious influence on cell growth or transcriptomes. Overall, this study sheds light on the high plasticity of rDNA organization and opens up opportunities for future rDNA engineering.

Building a eukaryotic chromosome arm by de novo design and synthesis.

The genome of an organism is inherited from its ancestor and continues to evolve over time, however, the extent to which the current version could be altered remains unknown. To probe the genome plasticity of Saccharomyces cerevisiae, here we replace the native left arm of chromosome XII (chrXIIL) with a linear artificial chromosome harboring small sets of reconstructed genes. We find that as few as 12 genes are sufficient for cell viability, whereas 25 genes are required to recover the partial fitness defects observed in the 12-gene strain. Next, we demonstrate that these genes can be reconstructed individually using synthetic regulatory sequences and recoded open-reading frames with a "one-amino-acid-one-codon" strategy to remain functional. Finally, a synthetic neochromsome with the reconstructed genes is assembled which could substitute chrXIIL for viability. Together, our work not only highlights the high plasticity of yeast genome, but also illustrates the possibility of making functional eukaryotic chromosomes from entirely artificial sequences.

Dissecting aneuploidy phenotypes by constructing Sc2.0 chromosome VII and SCRaMbLEing synthetic disomic yeast.

Aneuploidy compromises genomic stability, often leading to embryo inviability, and is frequently associated with tumorigenesis and aging. Different aneuploid chromosome stoichiometries lead to distinct transcriptomic and phenotypic changes, making it helpful to study aneuploidy in tightly controlled genetic backgrounds. By deploying the engineered SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution) system to the newly synthesized megabase Sc2.0 chromosome VII (synVII), we constructed a synthetic disomic yeast and screened hundreds of SCRaMbLEd derivatives with diverse chromosomal rearrangements. Phenotypic characterization and multi-omics analysis revealed that fitness defects associated with aneuploidy could be restored by (1) removing most of the chromosome content or (2) modifying specific regions in the duplicated chromosome. These findings indicate that both chromosome copy number and specific chromosomal regions contribute to the aneuploidy-related phenotypes, and the synthetic chromosome resource opens new paradigms in studying aneuploidy.

A DNA-based non-infectious replicon system to study SARS-CoV-2 RNA synthesis.

The coronavirus disease-2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has seriously affected public health around the world. In-depth studies on the pathogenic mechanisms of SARS-CoV-2 is urgently necessary for pandemic prevention. However, most laboratory studies on SARS-CoV-2 have to be carried out in bio-safety level 3 (BSL-3) laboratories, greatly restricting the progress of relevant experiments. In this study, we used a bacterial artificial chromosome (BAC) method to assemble a SARS-CoV-2 replication and transcription system in Vero E6 cells without virion envelope formation, thus avoiding the risk of coronavirus exposure. Furthermore, an improved real-time quantitative reverse transcription PCR (RT-qPCR) approach was used to distinguish the replication of full-length replicon RNAs and transcription of subgenomic RNAs (sgRNAs). Using the SARS-CoV-2 replicon, we demonstrated that the nucleocapsid (N) protein of SARS-CoV-2 facilitates the transcription of sgRNAs in the discontinuous synthesis process. Moreover, two high-frequency mutants of N protein, R203K and S194L, can obviously enhance the transcription level of the replicon, hinting that these mutations likely allow SARS-CoV-2 to spread and reproduce more quickly. In addition, remdesivir and chloroquine, two well-known drugs demonstrated to be effective against coronavirus in previous studies, also inhibited the transcription of our replicon, indicating the potential applications of this system in antiviral drug discovery. Overall, we developed a bio-safe and valuable replicon system of SARS-CoV-2 that is useful to study the mechanisms of viral RNA synthesis and has potential in novel antiviral drug screening.

Efficient de novo assembly and modification of large DNA fragments.

Synthetic genomics has provided new bottom-up platforms for the functional study of viral and microbial genomes. The construction of the large, gigabase (Gb)-sized genomes of higher organisms will deepen our understanding of genetic blueprints significantly. But for the synthesis and assembly of such large-scale genomes, the development of new or expanded methods is required. In this study, we develop an efficient pipeline for the construction of large DNA fragments sized 100 kilobases (kb) or above from scratches and describe an efficient method for "scar-free" engineering of the assembled sequences. Our method, therefore, should provide a standard framework for producing long DNA molecules, which are critical materials for synthetic genomics and metabolic engineering.

Chromosomal Rearrangements of Synthetic Yeast by SCRaMbLE.

Diversified genomes derived from chromosomal rearrangements are valuable materials for evolution. Naturally, chromosomal rearrangements occur at extremely low frequency to ensure genome stability. In the synthetic yeast genome project (Sc2.0), an inducible chromosome rearrangement system named Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) is built to produce chromosomal rearrangements such as deletion, duplication, inversion, and translocation at high efficiency. Here, we detail the method to activate SCRaMbLE in a synthetic strain, to analyze the SCRaMbLEd genome, and to dissect the causative rearrangements for a desired phenotype after SCRaMbLEing.

Use YeastFab to Construct Genetic Parts and Multicomponent Pathways for Metabolic Engineering.

Budding yeast, as a eukaryotic model organism, has well-defined genetic information and a highly efficient recombination system, making it a good host to produce exogenous chemicals. Since most metabolic pathways require multiple genes to function in coordination, it is usually laborious and time-consuming to construct a working pathway. To facilitate the construction and optimization of multicomponent exogenous pathways in yeast, we recently developed a method called YeastFab Assembly, which includes three steps: (1) make standard and reusable genetic parts, (2) construct transcription units from characterized parts, and (3) assemble a complete pathway. Here we describe a detailed protocol of this method.

Compacting a synthetic yeast chromosome arm.

BACKGROUND: Redundancy is a common feature of genomes, presumably to ensure robust growth under different and changing conditions. Genome compaction, removing sequences nonessential for given conditions, provides a novel way to understand the core principles of life. The synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE) system is a unique feature implanted in the synthetic yeast genome (Sc2.0), which is proposed as an effective tool for genome minimization. As the Sc2.0 project is nearing its completion, we have begun to explore the application of the SCRaMbLE system in genome compaction. RESULTS: We develop a method termed SCRaMbLE-based genome compaction (SGC) and demonstrate that a synthetic chromosome arm (synXIIL) can be efficiently reduced. The pre-introduced episomal essential gene array significantly enhances the compacting ability of SGC, not only by enabling the deletion of nonessential genes located in essential gene containing loxPsym units but also by allowing more chromosomal sequences to be removed in a single SGC process. Further compaction is achieved through iterative SGC, revealing that at least 39 out of 65 nonessential genes in synXIIL can be removed collectively without affecting cell viability at 30 °C in rich medium. Approximately 40% of the synthetic sequence, encoding 28 genes, is found to be dispensable for cell growth at 30 °C in rich medium and several genes whose functions are needed under specified conditions are identified. CONCLUSIONS: We develop iterative SGC with the aid of eArray as a generic yet effective tool to compact the synthetic yeast genome.

Genetic interaction mapping informs integrative structure determination of protein complexes.

Determining structures of protein complexes is crucial for understanding cellular functions. Here, we describe an integrative structure determination approach that relies on in vivo measurements of genetic interactions. We construct phenotypic profiles for point mutations crossed against gene deletions or exposed to environmental perturbations, followed by converting similarities between two profiles into an upper bound on the distance between the mutated residues. We determine the structure of the yeast histone H3-H4 complex based on ~500,000 genetic interactions of 350 mutants. We then apply the method to subunits Rpb1-Rpb2 of yeast RNA polymerase II and subunits RpoB-RpoC of bacterial RNA polymerase. The accuracy is comparable to that based on chemical cross-links; using restraints from both genetic interactions and cross-links further improves model accuracy and precision. The approach provides an efficient means to augment integrative structure determination with in vivo observations.

Whole genome engineering by synthesis.

Whole genome engineering is now feasible with the aid of genome editing and synthesis tools. Synthesizing a genome from scratch allows modifications of the genomic structure and function to an extent that was hitherto not possible, which will finally lead to new insights into the basic principles of life and enable valuable applications. With several recent genome synthesis projects as examples, the technical details to synthesize a genome and applications of synthetic genome are addressed in this perspective. A series of ongoing or future synthetic genomics projects, including the different genomes to be synthesized in GP-write, synthetic minimal genome, massively recoded genome, chimeric genome and synthetic genome with expanded genetic alphabet, are also discussed here with a special focus on theoretical and technical impediments in the design and synthesis process. Synthetic genomics will become a commonplace to engineer pathways and genomes according to arbitrary sets of design principles with the development of high-efficient, low-cost genome synthesis and assembly technologies.

Construction of Comprehensive Dosage-Matching Core Histone Mutant Libraries for Saccharomyces cerevisiae.

Saccharomyces cerevisiae contains two genes for each core histone, which are presented as pairs under the control of a divergent promoter, i.e., HHT1-HHF1, HHT2-HHF2, HTA1-HTB1 and HTA2-HTB2HHT1-HHF1, and HHT2-HHF2 encode histone H3 and H4 with identical amino acid sequences but under the control of differently regulated promoters. Previous mutagenesis studies were carried out by deleting one pair and mutating the other one. Here, we present the design and construction of three additional libraries covering HTA1-HTB1, HTA2-HTB2, and HHT1-HHF1 respectively. Together with the previously described library of HHT2-HHF2 mutants, a systematic and complete collection of mutants for each of the eight core S. cerevisiae histone genes becomes available. Each designed mutant was incorporated into the genome, generating three more corresponding libraries of yeast strains. We demonstrated that, although, under normal growth conditions, strains with single-copy integrated histone genes lacked phenotypes, in some growth conditions, growth deficiencies were observed. Specifically, we showed that addition of a second copy of the mutant histone gene could rescue the lethality in some previously known mutants that cannot survive with a single copy. This resource enables systematic studies of function of each nucleosome residue in plasmid, single-copy, and double-copy integrated formats.

Dissecting Nucleosome Function with a Comprehensive Histone H2A and H2B Mutant Library.

Using a comprehensive library of histone H2A and H2B mutants, we assessed the biological function of each amino acid residue involved in various stress conditions including exposure to different DNA damage-inducing reagents, different growth temperatures, and other chemicals. H2B N- and H2A C-termini were critical for maintaining nucleosome function and mutations in these regions led to pleiotropic phenotypes. Additionally, two screens were performed using this library, monitoring heterochromatin gene silencing and genome stability, to identify residues that could compromise normal function when mutated. Many distinctive regions within the nucleosome were revealed. Furthermore, we used the barcode sequencing (bar-seq) method to profile the mutant composition of many libraries in one high-throughput sequencing experiment, greatly reducing the labor and increasing the capacity. This study not only demonstrates the applications of the versatile histone library, but also reveals many previously unknown functions of histone H2A and H2B.