Coronaviruses have brought severe challenges to public health all over the world in the past 20years. SARS-CoV-2, the causative agent of the COVID-19 pandemic that has led to millions of deaths, belongs to the genus beta-coronavirus. Alpha- and beta-coronaviruses encode a unique protein, nonstructural protein 1 (Nsp1) that both suppresses host immune responses and reduces global gene expression levels in the host cells. As a key pathogenicity factor of coronaviruses, Nsp1 redirects the host translation machinery to increase synthesis of viral proteins. Through multiple mechanisms, coronaviruses impede host protein expression through Nsp1, while escaping inhibition to allow the translation of viral RNA. In this review, we discuss current data about suppression of the immune responses and inhibition of protein synthesis induced by coronavirus Nsp1, as well as the prospect of live-attenuated vaccine development with virulence-attenuated viruses with mutations in Nsp1.
Yeast whole genome sequencing (WGS) lacks end-to-end workflows that identify genetic engineering. Here we present Prymetime, a tool that assembles yeast plasmids and chromosomes and annotates genetic engineering sequences. It is a hybrid workflow—it uses short and long reads as inputs to perform separate linear and circular assembly steps. This structure is necessary to accurately resolve genetic engineering sequences in plasmids and the genome. We show this by assembling diverse engineered yeasts, in some cases revealing unintended deletions and integrations. Furthermore, the resulting whole genomes are high quality, although the underlying assembly software does not consistently resolve highly repetitive genome features. Finally, we assemble plasmids and genome integrations from metagenomic sequencing, even with 1 engineered cell in 1000. This work is a blueprint for building WGS workflows and establishes WGS-based identification of yeast genetic engineering.
Yeast genomes can be assembled from sequencing data, but genome integrations and episomal plasmids often fail to be resolved with accuracy, completeness, and contiguity. Resolution of these features is critical for many synthetic biology applications, including strain quality control and identifying engineering in unknown samples. Here, we report an integrated workflow, named Prymetime, that uses sequencing reads from inexpensive NGS platforms, assembly and error correction software, and a list of synthetic biology parts to achieve accurate whole genome sequences of yeasts with engineering annotated. To build the workflow, we first determined which sequencing methods and software packages returned an accurate, complete, and contiguous genome of an engineered S. cerevisiae strain with two similar plasmids and an integrated pathway. We then developed a sequence feature annotation step that labels synthetic biology parts from a standard list of yeast engineering sequences or from a custom sequence list. We validated the workflow by sequencing a collection of 15 engineered yeasts built from different parent S. cerevisiae and nonconventional yeast strains. We show that each integrated pathway and episomal plasmid can be correctly assembled and annotated, even in strains that have part repeats and multiple similar plasmids. Interestingly, Prymetime was able to identify deletions and unintended integrations that were subsequently confirmed by other methods. Furthermore, the whole genomes are accurate, complete, and contiguous. To illustrate this clearly, we used a publicly available S. cerevisiae CEN.PK113 reference genome and the accompanying reads to show that a Prymetime genome assembly is equivalent to the reference using several standard metrics. Finally, we used Prymetime to resequence the nonconventional yeasts Y. lipolytica Po1f and K. phaffii CBS 7435, producing an improved genome assembly for each strain. Thus, our workflow can achieve accurate, complete, and contiguous whole genome sequences of yeast strains before and after engineering. Therefore, Prymetime enables NGS-based strain quality control through assembly and identification of engineering features.Yet, applying WGS is a challenge because of the diversity of genetic backgrounds, the variety of engineering features, and the current scale of yeast strain engineering. Myriad laboratory strains of the baker's yeast Saccharomyces cerevisiae 9, 24, 25 and nonconventional yeasts like Yarrowia lipolytica 26-28 and Komagataella phaffii (formerly Pichia pastoris) 29, 30 are used to create yeast cell factories, so there are many potential genetic backgrounds. Methods of yeast engineering leave myriad sequence features behind, including standard plasmid sets with standard expression parts 31-34 , high efficiency transformation 35-37 , homologous recombination 10, 38-40 , gene knockouts using the Cre recombinase system 41 , and genome editing using RNAguided endonucleases 7,11,42,[44][45][46] . Furthermore, the scale of yeast engineering is increasing both in the...
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