Extensive testing is essential to break the transmission of SARS-CoV-2, which causes the ongoing COVID-19 pandemic. Here, we present a CRISPR-based diagnostic assay that is robust to viral genome ...mutations and temperature, produces results fast, can be applied directly on nasopharyngeal (NP) specimens without RNA purification, and incorporates a human internal control within the same reaction. Specifically, we show that the use of an engineered AsCas12a enzyme enables detection of wildtype and mutated SARS-CoV-2 and allows us to perform the detection step with loop-mediated isothermal amplification (LAMP) at 60-65 °C. We also find that the use of hybrid DNA-RNA guides increases the rate of reaction, enabling our test to be completed within 30 minutes. Utilizing clinical samples from 72 patients with COVID-19 infection and 57 healthy individuals, we demonstrate that our test exhibits a specificity and positive predictive value of 100% with a sensitivity of 50 and 1000 copies per reaction (or 2 and 40 copies per microliter) for purified RNA samples and unpurified NP specimens respectively.
We developed a computational framework to robustly identify RNA editing sites using transcriptome and genome deep-sequencing data from the same individual. As compared with previous methods, our ...approach identified a large number of Alu and non-Alu RNA editing sites with high specificity. We also found that editing of non-Alu sites appears to be dependent on nearby edited Alu sites, possibly through the locally formed double-stranded RNA structure.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Current methods for determining RNA structure with short-read sequencing cannot capture most differences between distinct transcript isoforms. Here we present RNA structure analysis using nanopore ...sequencing (PORE-cupine), which combines structure probing using chemical modifications with direct long-read RNA sequencing and machine learning to detect secondary structures in cellular RNAs. PORE-cupine also captures global structural features, such as RNA-binding-protein binding sites and reactivity differences at single-nucleotide variants. We show that shared sequences in different transcript isoforms of the same gene can fold into different structures, highlighting the importance of long-read sequencing for obtaining phase information. We also demonstrate that structural differences between transcript isoforms of the same gene lead to differences in translation efficiency. By revealing isoform-specific RNA structure, PORE-cupine will deepen understanding of the role of structures in controlling gene regulation.
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GEOZS, IJS, IMTLJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK, ZAGLJ
Li et al. (Research Articles, 1 July 2011, p. 53; published online 19 May 2011) reported widespread differences between the RNA and DNA sequences of the same human cells, including all 12 possible ...mismatch types. Before accepting such a fundamental claim, a deeper analysis of the sequencing data is required to discern true differences between RNA and DNA from potential artifacts.
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•A universal method was adopted to effectively identify biosensors for any metabolite.•Transcriptomic changes were analysed when S. cerevisiae was exposed to alcohols.•Promoters were ...identified that responded specifically to 1-butanol.•The promoter-based biosensors could distinguish varied 1-butanol production levels.
The present study aimed to develop a universal methodology for the discovery of biosensors sensitive to particular stresses or metabolites by using a transcriptome analysis, in order to address the need for in vivo biosensors to drive the engineering of microbial cell factories. The method was successfully applied to the discovery of 1-butanol sensors. In particular, the genome-wide transcriptome profiling of S. cerevisiae exposed to three similar short-chain alcohols, 1-butanol, 1-propanol, and ethanol, identified genes that were differentially expressed only under the treatment of 1-butanol. From these candidates, two promoters that responded specifically to 1-butanol were characterized in a dose-dependent manner and were used to distinguish differences in production levels among different 1-butanol producer strains. This strategy opens up new opportunities for the discovery of promoter-based biosensors and can potentially be used to identify biosensors for any metabolite that causes cellular transcriptomic changes.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Mammalian host cell lines are the preferred expression systems for the manufacture of complex therapeutics and recombinant proteins. However, the most utilized mammalian host systems, namely Chinese ...hamster ovary (CHO), Sp2/0 and NS0 mouse myeloma cells, can produce glycoproteins with non-human glycans that may potentially illicit immunogenic responses. Hence, we developed a fully human expression system based on HEK293 cells for the stable and high titer production of recombinant proteins by first knocking out GLUL (encoding glutamine synthetase) using CRISPR-Cas9 system. Expression vectors using human GLUL as selection marker were then generated, with recombinant human erythropoietin (EPO) as our model protein. Selection was performed using methionine sulfoximine (MSX) to select for high EPO expression cells. EPO production of up to 92700 U/mL of EPO as analyzed by ELISA or 696 mg/L by densitometry was demonstrated in a 2 L stirred-tank fed batch bioreactor. Mass spectrometry analysis revealed that N-glycosylation of the produced EPO was similar to endogenous human proteins and non-human glycan epitopes were not detected. Collectively, our results highlight the use of a human cellular expression system for the high titer and xenogeneic-free production of EPO and possibly other complex recombinant proteins.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) efficiently generate all embryonic cell lineages but rarely generate extraembryonic cell types. We found that microRNA
...deficiency expands the developmental potential of mouse pluripotent stem cells, yielding both embryonic and extraembryonic lineages and strongly inducing MuERV-L (MERVL) endogenous retroviruses, similar to what is seen with features of totipotent two-cell blastomeres.
restricts the acquisition of expanded cell fate potential in pluripotent stem cells, and it represses MERVL expression through transcriptional regulation, at least in part by targeting the transcription factor Gata2. Our studies reveal a complex molecular network that defines and restricts pluripotent developmental potential in cultured ESCs and iPSCs.
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The prokaryotic type II CRISPR/Cas9 system has been adapted to perform targeted genome editing in cells and model organisms. Here, we describe targeted gene deletion and replacement in human cells ...via the CRISPR/Cas9 system using two guide RNAs. The system effectively generated targeted deletions of varied length, regardless of the transcriptional status of the target gene. It is notable that targeted gene deletions generated via CRISPR/Cas9 and two guide RNAs resulted in the formation of correct junctions at high efficiency. Moreover, in the presence of a homology repair donor, the CRISPR/Cas9 system could guide precise gene replacement. Our results illustrate that the CRISPR/Cas9 system can be used to precisely and effectively generate targeted deletions or gene replacement in human cells, which will facilitate characterization of functional domains in protein-coding genes as well as noncoding regulatory sequences in animal genomes.
The Xenopus embryo has provided key insights into fate specification, the cell cycle, and other fundamental developmental and cellular processes, yet a comprehensive understanding of its ...transcriptome is lacking. Here, we used paired end RNA sequencing (RNA-seq) to explore the transcriptome of Xenopus tropicalis in 23 distinct developmental stages. We determined expression levels of all genes annotated in RefSeq and Ensembl and showed for the first time on a genome-wide scale that, despite a general state of transcriptional silence in the earliest stages of development, approximately 150 genes are transcribed prior to the midblastula transition. In addition, our splicing analysis uncovered more than 10,000 novel splice junctions at each stage and revealed that many known genes have additional unannotated isoforms. Furthermore, we used Cufflinks to reconstruct transcripts from our RNA-seq data and found that ∼13.5% of the final contigs are derived from novel transcribed regions, both within introns and in intergenic regions. We then developed a filtering pipeline to separate protein-coding transcripts from noncoding RNAs and identified a confident set of 6686 noncoding transcripts in 3859 genomic loci. Since the current reference genome, XenTro3, consists of hundreds of scaffolds instead of full chromosomes, we also performed de novo reconstruction of the transcriptome using Trinity and uncovered hundreds of transcripts that are missing from the genome. Collectively, our data will not only aid in completing the assembly of the Xenopus tropicalis genome but will also serve as a valuable resource for gene discovery and for unraveling the fundamental mechanisms of vertebrate embryogenesis.
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