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The Sc2.0 global consortium to design and construct a synthetic genome based on the Saccharomyces cerevisiae genome commenced in 2006, comprising 16 synthetic chromosomes and a new-to-nature tRNA neochromosome. In this paper we describe assembly and debugging of the 902,994-bp synthetic Saccharomyces cerevisiae chromosome synXVI of the Sc2.0 project. Application of the CRISPR D-BUGS protocol identified defective loci, which were modified to improve sporulation and recover wild-type like growth when grown on glycerol as a sole carbon source when grown at 37˚C. LoxPsym sites inserted downstream of dubious open reading frames impacted the 5' UTR of genes required for optimal growth and were identified as a systematic cause of defective growth. Based on lessons learned from analysis of Sc2.0 defects and synXVI, an in-silico redesign of the synXVI chromosome was performed, which can be used as a blueprint for future synthetic yeast genome designs. The in-silico redesign of synXVI includes reduced PCR tag frequency, modified chunk and megachunk termini, and adjustments to allocation of loxPsym sites and TAA stop codons to dubious ORFs. This redesign provides a roadmap into applications of Sc2.0 strategies in non-yeast organisms., Competing Interests: Competing interests: T.C.W. and A.C.C. are founders and shareholders of Number 8 Bio Pty Ltd. J.D.B. is a Founder of and consultant to Opentrons LabWorks/Neochromosome, Inc, and serves or served on the Scientific Advisory Board of the following: CZ Biohub New York, LLC, Logomix, Inc., Modern Meadow, Inc., Rome Therapeutics, Inc., SeaHub, Seattle, WA, Tessera Therapeutics, Inc. and the Wyss Institute. J.S.B. is a Founder of Neochromosome, Inc., and a consultant to Opentrons Labworks, Inc. L.A.M. is a Founder of Neochromosome, Inc., and an employee of Opentrons Labworks, Inc. The remaining authors declare no competing interests., (© 2025. The Author(s).)

Engineering proteins is a challenging task requiring the exploration of a vast design space. Traditionally, this is achieved using Directed Evolution (DE), which is a laborious process. Generative deep learning, instead, can learn biological features of functional proteins from sequence and structural datasets and return novel variants. However, most models do not generate thermodynamically stable proteins, thus leading to many non-functional variants. Here we propose a model called PRotein Engineering by Variational frEe eNergy approximaTion (PREVENT), which generates stable and functional variants by learning the sequence and thermodynamic landscape of a protein. We evaluate PREVENT by designing 40 variants of the conditionally essential E. coli phosphotransferase N-acetyl-L-glutamate kinase (EcNAGK). We find 85% of the variants to be functional, with 55% of them showing similar growth rate compared to the wildtype enzyme, despite harbouring up to 9 mutations. Our results support a new approach that can significantly accelerate protein engineering., Competing Interests: Competing interests: Giovanni Stracquadanio is Co-Founder of ZYTHERA, a startup of the University of Edinburgh using AI and engineering biology to develop enzyme replacement therapies. The other authors declare no competing interests., (© 2024. The Author(s).)

Engineering proteins with desired functions and biochemical properties is pivotal for biotechnology and drug discovery. While computational methods based on evolutionary information are reducing the experimental burden by designing targeted libraries of functional variants, they still have a low success rate when the desired protein has few or very remote homologous sequences. Here we propose an autoregressive model, called Temporal Dirichlet Variational Autoencoder (TDVAE), which exploits the mathematical properties of the Dirichlet distribution and temporal convolution to efficiently learn high-order information from a functionally related, possibly remotely similar, set of sequences. TDVAE is highly accurate in predicting the effects of amino acid mutations, while being significantly 90% smaller than the other state-of-the-art models. We then use TDVAE to design variants of the human alpha galactosidase enzymes as potential treatment for Fabry disease. Our model builds a library of diverse variants which retain sequence, biochemical and structural properties of the wildtype protein, suggesting they could be suitable for enzyme replacement therapy. Taken together, our results show the importance of accurate sequence modelling and the potential of autoregressive models as protein engineering and analysis tools., (© 2024. The Author(s).)

Biofoundries are automated high-throughput facilities specializing in the design, construction, and testing of engineered/synthetic DNA constructs (plasmids), often from genetic parts. A critical step of this process is assessing the fidelity of the assembled DNA construct to the desired design. Current methods utilized for this purpose are restriction digest or PCR followed by fragment analysis and sequencing. The Edinburgh Genome Foundry (EGF) has recently established a single-molecule sequencing quality control step using the Oxford Nanopore sequencing technology, along with a companion Nextflow pipeline and a Python package, to perform in-depth analysis and generate a detailed report. Our software enables researchers working with plasmids, including biofoundry scientists, to rapidly analyze and interpret sequencing data. In conclusion, we have created a laboratory and software protocol that validates assembled, cloned, or edited plasmids, using Nanopore long-reads, which can serve as a useful resource for the genetics, synthetic biology, and sequencing communities.

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., Competing Interests: Declaration of interests J.D.B. is a founder and director of CDI Labs, Inc.; a founder of and consultant to Neochromosome, Inc; and a founder, Scientific Advisory Board (SAB) member of, and consultant to ReOpen Diagnostics, LLC. J.D.B. also serves or served on the SABs of the following: Logomix, Inc.; Sangamo, Inc.; Modern Meadow, Inc.; Rome Therapeutics, Inc.; Sample6, Inc.; Tessera Therapeutics, Inc.; and the Wyss Institute. Y.A. is a Founder and CSO of Logomix, Inc., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The Sc2.0 project is building a eukaryotic synthetic genome from scratch. A major milestone has been achieved with all individual Sc2.0 chromosomes assembled. Here, we describe the consolidation of multiple synthetic chromosomes using advanced endoreduplication intercrossing with tRNA expression cassettes to generate a strain with 6.5 synthetic chromosomes. The 3D chromosome organization and transcript isoform profiles were evaluated using Hi-C and long-read direct RNA sequencing. We developed CRISPR Directed Biallelic URA3-assisted Genome Scan, or "CRISPR D-BUGS," to map phenotypic variants caused by specific designer modifications, known as "bugs." We first fine-mapped a bug in synthetic chromosome II (synII) and then discovered a combinatorial interaction associated with synIII and synX, revealing an unexpected genetic interaction that links transcriptional regulation, inositol metabolism, and tRNA Ser CGA abundance. Finally, to expedite consolidation, we employed chromosome substitution to incorporate the largest chromosome (synIV), thereby consolidating >50% of the Sc2.0 genome in one strain., Competing Interests: Declaration of interests J.D.B. is a founder and director of CDI Labs, Inc., a founder of and consultant to Neochromosome, Inc., and 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., Logomix Inc., Modern Meadow, Inc., Rome Therapeutics, Inc., Sample6, Inc., Tessera Therapeutics, Inc., and the Wyss Institute., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Pioneering advances in genome engineering, and specifically in genome writing, have revolutionized the field of synthetic biology, propelling us toward the creation of synthetic genomes. The Sc2.0 project aims to build the first fully synthetic eukaryotic organism by assembling the genome of Saccharomyces cerevisiae . With the completion of synthetic chromosome VIII ( synVIII ) described here, this goal is within reach. In addition to writing the yeast genome, we sought to manipulate an essential functional element: the point centromere. By relocating the native centromere sequence to various positions along chromosome VIII , we discovered that the minimal 118-bp CEN8 sequence is insufficient for conferring chromosomal stability at ectopic locations. Expanding the transplanted sequence to include a small segment (∼500 bp) of the CDEIII -proximal pericentromere improved chromosome stability, demonstrating that minimal centromeres display context-dependent functionality., Competing Interests: J.B. 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: Logomix, Inc., Modern Meadow, Inc., Rome Therapeutics, Inc., Sample6, Inc., Sangamo, Inc., Tessera Therapeutics, Inc., and the Wyss Institute. J.S.B. is a founder of and consultant to Neochromosome. G.S. is a consultant to Neochromosome Inc. and ZenithAI., (© 2023 The Authors.)

Chromosome-level design-build-test-learn cycles (chrDBTLs) allow systematic combinatorial reconfiguration of chromosomes with ease. Here, we established chrDBTL with a redesigned synthetic Saccharomyces cerevisiae chromosome XV , synXV . We designed and built synXV to harbor strategically inserted features, modified elements, and synonymously recoded genes throughout the chromosome. Based on the recoded chromosome, we developed a method to enable chrDBTL: CRISPR-Cas9-mediated mitotic recombination with endoreduplication (CRIMiRE). CRIMiRE allowed the creation of customized wild-type/synthetic combinations, accelerating genotype-phenotype mapping and synthetic chromosome redesign. We also leveraged synXV as a "build-to-learn" model organism for translation studies by ribosome profiling. We conducted a locus-to-locus comparison of ribosome occupancy between synXV and the wild-type chromosome, providing insight into the effects of codon changes and redesigned features on translation dynamics in vivo . Overall, we established synXV as a versatile reconfigurable system that advances chrDBTL for understanding biological mechanisms and engineering strains., Competing Interests: J.D.B. is a founder and director of CDI Labs, Inc.; a founder of and consultant to Neochromosome, Inc.; and a founder of, scientific advisory board member of, and consultant to ReOpen Diagnostics, LLC and serves or served on the scientific advisory boards of the following: Sangamo, Inc.; Logomix, Inc.; Modern Meadow, Inc.; Rome Therapeutics, Inc.; Sample6, Inc.; Tessera Therapeutics, Inc.; and the Wyss Institute., (© 2023 The Authors.)

We designed and synthesized synI , which is ∼21.6% shorter than native chrI , the smallest chromosome in Saccharomyces cerevisiae . SynI was designed for attachment to another synthetic chromosome due to concerns surrounding potential instability and karyotype imbalance and is now attached to synIII , yielding the first synthetic yeast fusion chromosome. Additional fusion chromosomes were constructed to study nuclear function. ChrIII-I and chrIX-III-I fusion chromosomes have twisted structures, which depend on silencing protein Sir3. As a smaller chromosome, chrI also faces special challenges in assuring meiotic crossovers required for efficient homolog disjunction. Centromere deletions into fusion chromosomes revealed opposing effects of core centromeres and pericentromeres in modulating deposition of the crossover-promoting protein Red1. These effects extend over 100 kb and promote disproportionate Red1 enrichment, and thus crossover potential, on small chromosomes like chrI . These findings reveal the power of synthetic genomics to uncover new biology and deconvolute complex biological systems., Competing Interests: J.D.B, L.A.M., and J.S.B. are founders of Neochromosome, Inc. J.D.B. is also a consultant of Neochromosome; a Founder and Director of CDI Labs, Inc,; a Founder of, Scientific Advisory Board member of, and consultant to ReOpen Diagnostics, LLC; and serves or served on the Scientific Advisory Board of the following: Logomix, Inc.; Sangamo, Inc.; Modern Meadow, Inc.; Rome Therapeutics, Inc.; Sample6, Inc.; Tessera Therapeutics, Inc.; and the Wyss Institute. N.A. is a synthetic biology specialist at Alagene., (© 2023 The Authors.)

We describe construction of the synthetic yeast chromosome XI ( synXI ) and reveal the effects of redesign at non-coding DNA elements. The 660-kb synthetic yeast genome project (Sc2.0) chromosome was assembled from synthesized DNA fragments before CRISPR-based methods were used in a process of bug discovery, redesign, and chromosome repair, including precise compaction of 200 kb of repeat sequence. Repaired defects were related to poor centromere function and mitochondrial health and were associated with modifications to non-coding regions. As part of the Sc2.0 design, loxPsym sequences for Cre-mediated recombination are inserted between most genes. Using the GAP1 locus from chromosome XI, we show that these sites can facilitate induced extrachromosomal circular DNA (eccDNA) formation, allowing direct study of the effects and propagation of these important molecules. Construction and characterization of synXI contributes to our understanding of non-coding DNA elements, provides a useful tool for eccDNA study, and will inform future synthetic genome design., Competing Interests: B.A.B. is a scientific advisory board (SAB) member of Eden Bio Ltd. T.E. is a consultant to Replay Holdings, LLC and SAB member of Modern Synthesis, Inc. J.D.B. is a founder and director of CDI Labs, Inc.; a founder of and consultant to Neochromosome, Inc.; a consultant to Opentrons Labworks, Inc.; a founder and SAB member of and consultant to ReOpen Diagnostics, LLC; and serves or served on the SAB of the following: Sangamo, Inc.; Modern Meadow, Inc.; Rome Therapeutics, Inc.; Sample6, Inc.; Tessera Therapeutics, Inc.; and the Wyss Institute. L.A.M. is a founder of Neochromosome, Inc. and an employee of Opentrons Labworks, Inc., (© 2023 The Author(s).)

Synthetic chromosome engineering is a complex process due to the need to identify and repair growth defects and deal with combinatorial gene essentiality when rearranging chromosomes. To alleviate these issues, we have demonstrated novel approaches for repairing and rearranging synthetic Saccharomyces cerevisiae genomes. We have designed, constructed, and restored wild-type fitness to a synthetic 753,096-bp version of S. cerevisiae chromosome XIV as part of the Synthetic Yeast Genome project. In parallel to the use of rational engineering approaches to restore wild-type fitness, we used adaptive laboratory evolution to generate a general growth-defect-suppressor rearrangement in the form of increased TAR1 copy number. We also extended the utility of the synthetic chromosome recombination and modification by loxP sym-mediated evolution (SCRaMbLE) system by engineering synthetic-wild-type tetraploid hybrid strains that buffer against essential gene loss, highlighting the plasticity of the S. cerevisiae genome in the presence of rational and non-rational modifications., Competing Interests: T.C.W. and A.C.C. are founders and shareholders of Number 8 Bio Pty Ltd. I.T.P. is an advisor of Number 8 Bio Pty Ltd. J.D.B. 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. J.S.B. is a founder of Neochromosome, Inc. and a consultant to Opentrons Labworks, Inc. L.A.M. is a founder of Neochromosome, Inc. and an employee of Opentrons Labworks, Inc., (© 2023 The Author(s).)

We describe the complete synthesis, assembly, debugging, and characterization of a synthetic 404,963 bp chromosome, synIX (synthetic chromosome IX ). Combined chromosome construction methods were used to synthesize and integrate its left arm ( synIXL ) into a strain containing previously described synIXR . We identified and resolved a bug affecting expression of EST3 , a crucial gene for telomerase function, producing a synIX strain with near wild-type fitness. To facilitate future synthetic chromosome consolidation and increase flexibility of chromosome transfer between distinct strains, we combined chromoduction, a method to transfer a whole chromosome between two strains, with conditional centromere destabilization to substitute a chromosome of interest for its native counterpart. Both steps of this chromosome substitution method were efficient. We observed that wild-type II tended to co-transfer with synIX and was co-destabilized with wild-type IX , suggesting a potential gene dosage compensation relationship between these chromosomes., Competing Interests: J.D.B. 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: Logomix, Inc., Sangamo, Inc., Modern Meadow, Inc., Rome Therapeutics, Inc., Sample6, Inc., Tessera Therapeutics, Inc., and the Wyss Institute. J.S.B. is a founder of Neochromosome, Inc., consultant to Opentrons Labworks, Inc., and advisor to Reflexion Pharmaceuticals, Inc., (© 2023 The Authors.)

Rapid engineering of biological systems is currently hindered by limited integration of manufacturing constraints into the design process, ultimately reducing the yield of many synthetic biology workflows. Here we tackle DNA engineering as a multi-objective optimization problem aiming at finding the best tradeoff between design requirements and manufacturing constraints. We developed a new open-source algorithm for DNA engineering, called Multi-Objective Optimisation algorithm for DNA Design and Assembly, available as a Python and Anaconda package, as well as a Docker image. Experimental results show that our method provides near-optimal constructs and scales linearly with design complexity, effectively paving the way to rational engineering of DNA molecules from genes to genomes., (© The Author(s) 2021. Published by Oxford University Press.)

Decades of research have shown that rare highly penetrant mutations can promote tumorigenesis, but it is still unclear whether variants observed at high-frequency in the broader population could modulate the risk of developing cancer. Genome-wide Association Studies (GWAS) have generated a wealth of data linking single nucleotide polymorphisms (SNPs) to increased cancer risk, but the effect of these mutations are usually subtle, leaving most of cancer heritability unexplained. Understanding the role of high-frequency mutations in cancer can provide new intervention points for early diagnostics, patient stratification and treatment in malignancies with high prevalence, such as breast cancer. Here we review state-of-the-art methods to study cancer heritability using GWAS data and provide an updated map of breast cancer susceptibility loci at the SNP and gene level., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Height and other anthropometric measures are consistently found to associate with differential cancer risk. However, both genetic and mechanistic insights into these epidemiological associations are notably lacking. Conversely, inherited genetic variants in tumour suppressors and oncogenes increase cancer risk, but little is known about their influence on anthropometric traits., Methods: By integrating inherited and somatic cancer genetic data from the Genome-Wide Association Study Catalog, expression Quantitative Trait Loci databases and the Cancer Gene Census, we identify SNPs that associate with different cancer types and differential gene expression in at least one tissue type, and explore the potential pleiotropic associations of these SNPs with anthropometric traits through SNP-wise association in a cohort of 500,000 individuals., Results: We identify three regulatory SNPs for three important cancer genes, FANCA, MAP3K1 and TP53 that associate with both anthropometric traits and cancer risk. Of particular interest, we identify a previously unrecognised strong association between the rs78378222[C] SNP in the 3' untranslated region (3'-UTR) of TP53 and both increased risk for developing non-melanomatous skin cancer (OR=1.36 (95% 1.31 to 1.41), adjusted p=7.62E -63 ), brain malignancy (OR=3.12 (2.22 to 4.37), adjusted p=1.43E -12 ) and increased standing height (adjusted p=2.18E -24 , beta=0.073±0.007), lean body mass (adjusted p=8.34E -37 , beta=0.073±0.005) and basal metabolic rate (adjusted p=1.13E -31 , beta=0.076±0.006), thus offering a novel genetic link between these anthropometric traits and cancer risk., Conclusion: Our results clearly demonstrate that heritable variants in key cancer genes can associate with both differential cancer risk and anthropometric traits in the general population, thereby lending support for a genetic basis for linking these human phenotypes., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Genome-wide association studies (GWAS) have found hundreds of single-nucleotide polymorphisms (SNP) associated with increased risk of cancer. However, the amount of heritable risk explained by SNPs is limited, leaving most of the cancer heritability unexplained. Tumor sequencing projects have shown that causal mutations are enriched in genic regions. We hypothesized that SNPs located in protein coding genes and nearby regulatory regions could explain a significant proportion of the heritable risk of cancer. To perform gene-level heritability analysis, we developed a new method, called Bayesian Gene Heritability Analysis (BAGHERA), to estimate the heritability explained by all genotyped SNPs and by those located in genic regions using GWAS summary statistics. BAGHERA was specifically designed for low heritability traits such as cancer and provides robust heritability estimates under different genetic architectures. BAGHERA-based analysis of 38 cancers reported in the UK Biobank showed that SNPs explain at least 10% of the heritable risk for 14 of them, including late onset malignancies. We then identified 1,146 genes, called cancer heritability genes (CHG), explaining a significant proportion of cancer heritability. CHGs were involved in hallmark processes controlling the transformation from normal to cancerous cells. Importantly, 60 of them also harbored somatic driver mutations, and 27 are tumor suppressors. Our results suggest that germline and somatic mutation information could be exploited to identify subgroups of individuals at higher risk of cancer in the broader population and could prove useful to establish strategies for early detection and cancer surveillance. SIGNIFICANCE: This study describes a new statistical method to identify genes associated with cancer heritability in the broader population, creating a map of the heritable cancer genome with gene-level resolution. See related commentary by Bader, p. 2586 ., (©2021 American Association for Cancer Research.)

Insights into oncogenesis derived from cancer susceptibility loci (SNP) hold the potential to facilitate better cancer management and treatment through precision oncology. However, therapeutic insights have thus far been limited by our current lack of understanding regarding both interactions of these loci with somatic cancer driver mutations and their influence on tumorigenesis. For example, although both germline and somatic genetic variation to the p53 tumor suppressor pathway are known to promote tumorigenesis, little is known about the extent to which such variants cooperate to alter pathway activity. Here we hypothesize that cancer risk-associated germline variants interact with somatic TP53 mutational status to modify cancer risk, progression, and response to therapy. Focusing on a cancer risk SNP (rs78378222) with a well-documented ability to directly influence p53 activity as well as integration of germline datasets relating to cancer susceptibility with tumor data capturing somatically-acquired genetic variation provided supportive evidence for this hypothesis. Integration of germline and somatic genetic data enabled identification of a novel entry point for therapeutic manipulation of p53 activities. A cluster of cancer risk SNPs resulted in increased expression of prosurvival p53 target gene KITLG and attenuation of p53-mediated responses to genotoxic therapies, which were reversed by pharmacologic inhibition of the prosurvival c-KIT signal. Together, our results offer evidence of how cancer susceptibility SNPs can interact with cancer driver genes to affect cancer progression and identify novel combinatorial therapies. SIGNIFICANCE: These results offer evidence of how cancer susceptibility SNPs can interact with cancer driver genes to affect cancer progression and present novel therapeutic targets., (©2021 American Association for Cancer Research.)

Synthetic biology aims at engineering biological systems, ranging from genes to entire genomes. The emerging field of synthetic genomics provides new tools to address questions and tackle challenges in biology and biotechnology impossible to address with current methods. Chromosome scale engineering requires computational tools and workflows to streamline designing, building and verifying long DNA molecules. While a systematic and generic genome design workflow does not exist, here we outline chromosome assembly and verification operations that are at the foundation of every genome scale engineering efforts.

Background: Gene and protein interaction experiments provide unique opportunities to study the molecular wiring of a cell. Integrating high-throughput functional genomics data with this information can help identifying networks associated with complex diseases and phenotypes., Results: Here we introduce an integrated statistical framework to test network properties of single and multiple genesets under different interaction models. We implemented this framework as an open-source software, called Python Geneset Network Analysis (PyGNA). Our software is designed for easy integration into existing analysis pipelines and to generate high quality figures and reports. We also developed PyGNA to take advantage of multi-core systems to generate calibrated null distributions on large datasets. We then present the results of extensive benchmarking of the tests implemented in PyGNA and a use case inspired by RNA sequencing data analysis, showing how PyGNA can be easily integrated to study biological networks. PyGNA is available at http://github.com/stracquadaniolab/pygna and can be easily installed using the PyPi or Anaconda package managers, and Docker., Conclusions: We present a tool for network-aware geneset analysis. PyGNA can either be readily used and easily integrated into existing high-performance data analysis pipelines or as a Python package to implement new tests and analyses. With the increasing availability of population-scale omic data, PyGNA provides a viable approach for large scale geneset network analysis.

IL-17 mediates immune protection from fungi and bacteria, as well as it promotes autoimmune pathologies. However, the regulation of the signal transduction from the IL-17 receptor (IL-17R) remained elusive. We developed a novel mass spectrometry-based approach to identify components of the IL-17R complex followed by analysis of their roles using reverse genetics. Besides the identification of linear ubiquitin chain assembly complex (LUBAC) as an important signal transducing component of IL-17R, we established that IL-17 signaling is regulated by a robust negative feedback loop mediated by TBK1 and IKKε. These kinases terminate IL-17 signaling by phosphorylating the adaptor ACT1 leading to the release of the essential ubiquitin ligase TRAF6 from the complex. NEMO recruits both kinases to the IL-17R complex, documenting that NEMO has an unprecedented negative function in IL-17 signaling, distinct from its role in NF-κB activation. Our study provides a comprehensive view of the molecular events of the IL-17 signal transduction and its regulation., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

Perfect matching of an assembled physical sequence to a specified designed sequence is crucial to verify design principles in genome synthesis. We designed and de novo synthesized 536,024-base pair chromosome synV in the "Build-A-Genome China" course. We corrected an initial isolate of synV to perfectly match the designed sequence using integrative cotransformation and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated editing in 22 steps; synV strains exhibit high fitness under a variety of culture conditions, compared with that of wild-type V strains. A ring synV derivative was constructed, which is fully functional in Saccharomyces cerevisiae under all conditions tested and exhibits lower spore viability during meiosis. Ring synV chromosome can extends Sc2.0 design principles and provides a model with which to study genomic rearrangement, ring chromosome evolution, and human ring chromosome disorders., (Copyright © 2017, American Association for the Advancement of Science.)

Here, we report the successful design, construction, and characterization of a 770-kilobase synthetic yeast chromosome II (synII). Our study incorporates characterization at multiple levels-including phenomics, transcriptomics, proteomics, chromosome segregation, and replication analysis-to provide a thorough and comprehensive analysis of a synthetic chromosome. Our Trans-Omics analyses reveal a modest but potentially relevant pervasive up-regulation of translational machinery observed in synII, mainly caused by the deletion of 13 transfer RNAs. By both complementation assays and SCRaMbLE (synthetic chromosome rearrangement and modification by loxP -mediated evolution), we targeted and debugged the origin of a growth defect at 37°C in glycerol medium, which is related to misregulation of the high-osmolarity glycerol response. Despite the subtle differences, the synII strain shows highly consistent biological processes comparable to the native strain., (Copyright © 2017, American Association for the Advancement of Science.)

We describe complete design of a synthetic eukaryotic genome, Sc2.0, a highly modified Saccharomyces cerevisiae genome reduced in size by nearly 8%, with 1.1 megabases of the synthetic genome deleted, inserted, or altered. Sc2.0 chromosome design was implemented with BioStudio, an open-source framework developed for eukaryotic genome design, which coordinates design modifications from nucleotide to genome scales and enforces version control to systematically track edits. To achieve complete Sc2.0 genome synthesis, individual synthetic chromosomes built by Sc2.0 Consortium teams around the world will be consolidated into a single strain by "endoreduplication intercross." Chemically synthesized genomes like Sc2.0 are fully customizable and allow experimentalists to ask otherwise intractable questions about chromosome structure, function, and evolution with a bottom-up design strategy., (Copyright © 2017, American Association for the Advancement of Science.)

Debugging a genome sequence is imperative for successfully building a synthetic genome. As part of the effort to build a designer eukaryotic genome, yeast synthetic chromosome X (synX), designed as 707,459 base pairs, was synthesized chemically. SynX exhibited good fitness under a wide variety of conditions. A highly efficient mapping strategy called pooled PCRTag mapping (PoPM), which can be generalized to any watermarked synthetic chromosome, was developed to identify genetic alterations that affect cell fitness ("bugs"). A series of bugs were corrected that included a large region bearing complex amplifications, a growth defect mapping to a recoded sequence in FIP1 , and a loxPsym site affecting promoter function of ATP2 PoPM is a powerful tool for synthetic yeast genome debugging and an efficient strategy for phenotype-genotype mapping., (Copyright © 2017, American Association for the Advancement of Science.)

We describe design, rapid assembly, and characterization of synthetic yeast Sc2.0 chromosome VI (synVI). A mitochondrial defect in the synVI strain mapped to synonymous coding changes within PRE4 ( YFR050C ), encoding an essential proteasome subunit; Sc2.0 coding changes reduced Pre4 protein accumulation by half. Completing Sc2.0 specifies consolidation of 16 synthetic chromosomes into a single strain. We investigated phenotypic, transcriptional, and proteomewide consequences of Sc2.0 chromosome consolidation in poly-synthetic strains. Another "bug" was discovered through proteomic analysis, associated with alteration of the HIS2 transcription start due to transfer RNA deletion and loxPsym site insertion. Despite extensive genetic alterations across 6% of the genome, no major global changes were detected in the poly-synthetic strain "omics" analyses. This work sets the stage for completion of a designer, synthetic eukaryotic genome., (Copyright © 2017, American Association for the Advancement of Science.)