Biology subjects

Lauer, S.

Publications and source records attributed to Lauer, S..

5 recordsLinked to original sources

bioRxiv2022

Synthetic yeast chromosome XI design enables extrachromosomal circular DNA formation on demand

We describe construction of the 660 kilobase synthetic yeast chromosome XI (synXI) and reveal how synthetic redesign of non-coding DNA elements impact the cell. To aid construction from synthesized 5 to 10 kilobase DNA fragments, we implemented CRISPR-based methods for synthetic crossovers in vivo and used these methods in an extensive process of bug discovery, redesign and chromosome repair, including for the precise removal of 200 kilobases of unexpected repeated sequence. In synXI, the underlying causes of several fitness defects were identified as modifications to non-coding DNA, including defects related to centromere function and mitochondrial activity that were subsequently corrected. As part of synthetic yeast chromosome design, loxPsym sequences for Cre-mediated recombination are inserted between most genes. Using the GAP1 locus from chromosome XI, we show here that targeted insertion of these sites can be used to create extrachromosomal circular DNA on demand, allowing direct study of the effects and propagation of these important molecules. Construction and characterization of synXI has uncovered effects of non-coding and extrachromosomal circular DNA, contributing to better understanding of these elements and informing future synthetic genome design.

synthetic biology↗

bioRxiv2022

Episomal editing of synthetic constructs in yeast using CRISPR

Use of synthetic genomics to design and build "big" DNA has revolutionized our ability to answer fundamental biological questions by employing a bottom-up approach. S. cerevisiae, or budding yeast, has become the major platform to assemble large synthetic constructs thanks to its powerful homologous recombination machinery and the availability of well-established molecular biology techniques. However, efficiently and precisely introducing designer variations to episomal assemblies remains challenging. Here, we describe CRISPR Engineering of EPisomes in Yeast, or CREEPY, for rapid engineering of mammalian DNA constructs larger than 100 kb. We demonstrate that editing of circular episomes presents unique challenges compared to modifying native yeast chromosomes with CRISPR. After optimizing CREEPY for episomal editing, we achieve efficient simplex and multiplex editing as demonstrated by engineering a mouse Sox2-harboring episome.

synthetic biology↗

bioRxiv2022

Structure and mechanism of a novel cytomegaloviral DCAF mediating interferon antagonism

Human cytomegalovirus (CMV) is a highly relevant and ubiquitously distributed human pathogen. Its rodent counterparts such as mouse and rat CMV serve as common infection models. Here, we conducted the first global proteome profiling of rat CMV-infected cells and uncovered a pronounced loss of the transcription factor STAT2, which is crucial for interferon signalling. Deletion mutagenesis documented that STAT2 is targeted by the viral protein E27. Cellular and in vitro analyses showed that E27 exploits host-derived Cullin4-RING ubiquitin ligases (CRL4) to induce poly-ubiquitylation and proteasomal degradation of STAT2. A cryo-electron microscopic structure determination revealed how E27 mimics molecular surface properties of cellular CRL4 substrate receptors called DDB1- and Cullin4-associated factors (DCAFs) to displace them from the catalytic core of CRL4. Moreover, structural analyses elucidated the mechanism of STAT2 recruitment and indicate that E27-binding additionally disturbs STAT2-dependent interferon signalling by occupying its IRF9 binding interface. For the first time, these data provide structural insights into cytomegalovirus-encoded interferon antagonism and establish an atomic model for STAT2 counteraction by CRL4 misappropriation with important implications for viral immune evasion.

biochemistry↗

bioRxiv2022

Debugging and consolidating multiple synthetic chromosomes reveals combinatorial genetic interactions

The Sc2.0 project is building a eukaryotic synthetic genome from scratch, incorporating thousands of designer features. A major milestone has been achieved with the assembly of all individual Sc2.0 chromosomes. Here, we describe the consolidation of multiple synthetic chromosomes using endoreduplication intercross to generate a strain with 6.5 synthetic chromosomes. Genome-wide chromosome conformation capture and long-read direct RNA sequencing were performed on this strain to evaluate the effects of designer modifications, such as loxPsym site insertion, tRNA relocation, and intron deletion, on 3D chromosome organization and transcript isoform profiles. To precisely map "bugs", we developed a method, CRISPR Directed Biallelic URA3-assisted Genome Scan, or "CRISPR D-BUGS", exploiting directed mitotic recombination in heterozygous diploids. Using this method, we first fine-mapped a synII defect resulting from two loxPsym sites in the 3 UTR of SHM1. This approach was also used to map a combinatorial bug associated with synIII and synX, revealing a highly unexpected genetic interaction that links transcriptional regulation, inositol metabolism and tRNASerCGA abundance. "Starvation" for tRNASerCGA leads to insufficient levels of the key positive inositol biosynthesis regulator, Swi3, which contains tandem UCG codons. Finally, to expedite consolidation, we employed a new method, chromosome swapping, to incorporate the largest chromosome (synIV), thereby consolidating more than half of the Sc2.0 genome in a single strain.

genomics↗

bioRxiv2018

Single-cell copy number variant detection reveals the dynamics and diversity of adaptation

Copy number variants (CNVs) are a pervasive, but understudied source of genetic variation and evolutionary potential. Long-term evolution experiments in chemostats provide an ideal system for studying the molecular processes underlying CNV formation and the temporal dynamics of de novo CNVs. Here, we developed a fluorescent reporter to monitor gene amplifications and deletions at a specific locus with single-cell resolution. Using a CNV reporter in nitrogen-limited chemostats, we find that GAP1 CNVs are repeatedly generated and selected during the early stages of adaptive evolution resulting in predictable dynamics of CNV selection. However, subsequent diversification of populations defines a second phase of evolutionary dynamics that cannot be predicted. Using whole genome sequencing, we identified a variety of GAP1 CNVs that vary in size and copy number. Despite GAP1s proximity to tandem repeats that facilitate intrachromosomal recombination, we find that non-allelic homologous recombination (NAHR) between flanking tandem repeats occurs infrequently. Rather, breakpoint characterization revealed that for at least 50% of GAP1 CNVs, origin-dependent inverted-repeat amplification (ODIRA), a DNA replication mediated process, is the likely mechanism. We also find evidence that ODIRA generates DUR3 CNVs, indicating that it may be a common mechanism of gene amplification. We combined the CNV reporter with barcode lineage tracking and found that 103-104 independent CNV-containing lineages initially compete within populations, which results in extreme clonal interference. Our study introduces a novel means of studying CNVs in heterogeneous cell populations and provides insight into the underlying dynamics of CNVs in evolution.

evolutionary biology↗