scholarly journals PEAR: a flexible fluorescent reporter for the identification and enrichment of successfully prime edited cells

2021 ◽  
Author(s):  
Dorottya A. Simon ◽  
András Tálas ◽  
Péter I. Kulcsár ◽  
Ervin Welker
Keyword(s):  
Single Cells ◽  
Full Length ◽  
Target Sequence ◽  
Gene Engineering ◽  
Fluorescent Reporter ◽  
Background Fluorescence ◽  
Sequence Variations ◽  
Fundamental Research ◽  
Editing Activity

ABSTRACTPrime editing is a recently developed gene engineering tool that allows the introduction of short insertions, deletions or substitutions into the genome. However, the efficiency of prime editing, generally reaching around 10-30% editing, has not resembled its versatility. Here, Prime Editor Activity Reporter (PEAR), a sensitive fluorescent tool is introduced for the identification of single cells with prime editing activity. Possessing no background fluorescence, PEAR specifically reports on prime editing events in individual cells. By design, it ensures unrestricted flexibility for sequence variations in the full length of the target sequence. The application of PEAR as an enrichment marker of prime editing can increase the edited population by up to 70% and alleviate the burden of the otherwise time and labour consuming process of cloning of the correctly edited cells, therefore considerably improving the applicability of prime editing in fundamental research and biotechnological uses.

scholarly journals Two methods for full-length RNA sequencing for low quantities of cells and single cells

2012 ◽  
Vol 110 (2) ◽  
pp. 594-599 ◽  
Author(s):  
X. Pan ◽  
R. E. Durrett ◽  
H. Zhu ◽  
Y. Tanaka ◽  
Y. Li ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Single Cells ◽  
Full Length

scholarly journals A Reverse Genetics System for Zika Virus Based on a Simple Molecular Cloning Strategy

Viruses ◽  
2018 ◽  
Vol 10 (7) ◽  
pp. 368 ◽  
Author(s):  
Maximilian Münster ◽  
Anna Płaszczyca ◽  
Mirko Cortese ◽  
Christopher Neufeldt ◽  
Sarah Goellner ◽  
...  
Keyword(s):  
Zika Virus ◽  
In Silico ◽  
Reverse Genetics ◽  
Molecular Mechanisms ◽  
Full Length ◽  
Cdna Clones ◽  
In Silico Prediction ◽  
Fluorescent Reporter ◽  
Full Length Cdna ◽  
Spurious Transcription

The Zika virus (ZIKV) has recently attracted major research interest as infection was unexpectedly associated with neurological manifestations in developing foetuses and with Guillain-Barré syndrome in infected adults. Understanding the underlying molecular mechanisms requires reverse genetic systems, which allow manipulation of infectious cDNA clones at will. In the case of flaviviruses, to which ZIKV belongs, several reports have indicated that the construction of full-length cDNA clones is difficult due to toxicity during plasmid amplification in Escherichia coli. Toxicity of flaviviral cDNAs has been linked to the activity of cryptic prokaryotic promoters within the region encoding the structural proteins leading to spurious transcription and expression of toxic viral proteins. Here, we employ an approach based on in silico prediction and mutational silencing of putative promoters to generate full-length cDNA clones of the historical MR766 strain and the contemporary French Polynesian strain H/PF/2013 of ZIKV. While for both strains construction of full-length cDNA clones has failed in the past, we show that our approach generates cDNA clones that are stable on single bacterial plasmids and give rise to infectious viruses with properties similar to those generated by other more complex assembly strategies. Further, we generate luciferase and fluorescent reporter viruses as well as sub-genomic replicons that are fully functional and suitable for various research and drug screening applications. Taken together, this study confirms that in silico prediction and silencing of cryptic prokaryotic promoters is an efficient strategy to generate full-length cDNA clones of flaviviruses and reports novel tools that will facilitate research on ZIKV biology and development of antiviral strategies.

scholarly journals The flexible C terminus of the rotavirus non-structural protein NSP4 is an important determinant of its biological properties

2008 ◽  
Vol 89 (6) ◽  
pp. 1485-1496 ◽  
Author(s):  
Deepa Rajasekaran ◽  
Narayan P. Sastri ◽  
Jagannath R. Marathahalli ◽  
Shanthinath S. Indi ◽  
Kiranmayee Pamidimukkala ◽  
...  
Keyword(s):  
Protein A ◽  
Biological Properties ◽  
Full Length ◽  
Structural Protein ◽  
Newborn Mice ◽  
Suckling Mice ◽  
C Terminus ◽  
Tht Fluorescence ◽  
Sequence Variations ◽  
Optimal Functions

The rotavirus non-structural protein NSP4 functions as the viral enterotoxin and intracellular receptor for the double-layered particles (DLP). The full-length protein cannot be expressed and/or purified to homogeneity from bacterial or insect cells. However, a bacterially expressed and purified mutant lacking the N-terminal 72 aa (ΔN72) was recently obtained from strains Hg18 and SA11 exhibiting approximately 17–20-, 150–200- and 13166–15800-fold lower DD50 (50% diarrhoea-inducing dose) values in suckling mice compared with that reported for the partially pure, full-length protein, a C-terminal M175I mutant and a synthetic peptide comprising aa 114–135, respectively, suggesting the requirement for a unique conformation for optimal functions of the purified protein. The stretch of approximately 40 aa from the C terminus of the cytoplasmic tail of the endoplasmic reticulum-anchored NSP4 is highly flexible and exhibits high sequence variation compared with the other regions, the significance of which in diarrhoea induction remain unresolved. Here, it was shown that every amino acid substitution or deletion in the flexible C terminus resulted in altered conformation, multimerization, trypsin resistance and thioflavin T (ThT) binding, and affected DLP binding and the diarrhoea-inducing ability of the highly diarrhoeagenic SA11 and Hg18 ΔN72 in suckling mice. These studies further revealed that high ThT fluorescence correlated with efficient diarrhoea induction, suggesting the importance of an optimal ThT-recognizable conformation in diarrhoea induction by purified NSP4. These results based on biological properties provide a possible conformational basis for understanding the influence of primary sequence variations on diarrhoea induction in newborn mice by purified NSP4s that cannot be explained by extensive sequence analyses.

scholarly journals Non-canonical Features of Pentatricopeptide Repeat Protein-Facilitated RNA Editing in Arabidopsis Chloroplasts

2019 ◽  
Author(s):  
Yueming Kelly Sun ◽  
Bernard Gutmann ◽  
Ian Small
Keyword(s):  
Rna Editing ◽  
Plant Mitochondria ◽  
Pentatricopeptide Repeat ◽  
Site Specific ◽  
Ppr Protein ◽  
Sequence Variations ◽  
Ppr Proteins ◽  
Editing Activity ◽  
Chloroplast Rna ◽  
Plant Organelles

AbstractCytosine (C) to uracil (U) RNA editing in plant mitochondria and chloroplasts is facilitated by site-specific pentatricopeptide repeat (PPR) editing factors. PPR editing factors contain multiple types of PPR motifs, and PPR motifs of the same type also show sequence variations. Therefore, no PPR motifs are invariant within a PPR protein or between different PPR proteins. This work evaluates the functional diversity of PPR motifs in CHLOROPLAST RNA EDITING FACTOR 3 (CREF3). The results indicate that previously overlooked features of PPR editing factors could also contribute to RNA editing activity. In particular, the N-terminal degenerated PPR motifs and the two L1-type PPR motifs in CREF3 are functionally indispensable. Furthermore, PPR motifs of the same type in CREF3 are not interchangeable. These non-canonical features of CREF3 have important implications on the understanding of PPR-facilitated RNA editing in plant organelles.

scholarly journals Poor concordance of floxed sequence recombination in single neural stem cells: Implications for cell autonomous studies

2019 ◽  
Author(s):  
Tyler Joseph Dause ◽  
Elizabeth Diana Kirby
Keyword(s):  
Gene Function ◽  
Target Gene ◽  
Single Cells ◽  
Gene Recombination ◽  
Cell Level ◽  
Fluorescent Reporter ◽  
Site Specific ◽  
Poor Predictor ◽  
Adult Cell ◽  
Stem And Progenitor Cells

SummaryTo manipulate target gene function in specific adult cell populations, tamoxifen-dependent CreERT2 is widely used to drive inducible, site-specific recombination of LoxP flanked sequences. In studies of cell autonomous target gene function, it is common practice to combine these CreERT2-lox systems with a ubiquitously-expressed stop-floxed fluorescent reporter gene to identify single cells supposedly undergoing target gene recombination. Here, we studied the reliability of using Cre-induced recombination of one gene to predict recombination in another gene at the single cell level in adult hippocampal neural stem and progenitor cells. Using two separate stop-floxed reporters plus a Nestin promoter-driven CreERT2, we found that, in individual cells, expression of one reporter was a poor predictor of expression of the other. These findings imply that use of stop-floxed reporters to investigate cell autonomous gene function is likely to lead to false conclusions because recombination in separate genes shows poor concordance in individual cells.

scholarly journals Comprehensive characterization of single cell full-length isoforms in human and mouse with long-read sequencing

2020 ◽  
Author(s):  
Luyi Tian ◽  
Jafar S. Jabbari ◽  
Rachel Thijssen ◽  
Quentin Gouil ◽  
Shanika L. Amarasinghe ◽  
...  
Keyword(s):  
Data Integration ◽  
Single Cell ◽  
Ribosome Biogenesis ◽  
Single Cells ◽  
Transcript Level ◽  
Full Length ◽  
Alternative Transcript ◽  
Long Read ◽  
Comprehensive Characterization

AbstractAlternative splicing shapes the phenotype of cells in development and disease. Long-read RNA-sequencing recovers full-length transcripts but has limited throughput at the single-cell level. Here we developed single-cell full-length transcript sequencing by sampling (FLT-seq), together with the computational pipeline FLAMES to overcome these issues and perform isoform discovery and quantification, splicing analysis and mutation detection in single cells. With FLT-seq and FLAMES, we performed the first comprehensive characterization of the full-length isoform landscape in single cells of different types and species and identified thousands of unannotated isoforms. We found conserved functional modules that were enriched for alternative transcript usage in different cell populations, including ribosome biogenesis and mRNA splicing. Analysis at the transcript-level allowed data integration with scATAC-seq on individual promoters, improved correlation with protein expression data and linked mutations known to confer drug resistance to transcriptome heterogeneity. Our methods reveal previously unseen isoform complexity and provide a better framework for multi-omics data integration.

scholarly journals BE-FLARE: a fluorescent reporter of base editing activity reveals editing characteristics of APOBEC3A and APOBEC3B

BMC Biology ◽  
2018 ◽  
Vol 16 (1) ◽  
Author(s):  
Matthew A. Coelho ◽  
Songyuan Li ◽  
Luna Simona Pane ◽  
Mike Firth ◽  
Giovanni Ciotta ◽  
...  
Keyword(s):  
Fluorescent Reporter ◽  
Base Editing ◽  
Editing Activity

scholarly journals Microfluidic platform enables live-cell imaging of signaling and transcription combined with multiplexed secretion measurements in the same single cells

2019 ◽  
Vol 11 (4) ◽  
pp. 142-153 ◽  
Author(s):  
Ramesh Ramji ◽  
Amanda F Alexander ◽  
Andrés R Muñoz-Rojas ◽  
Laura N Kellman ◽  
Kathryn Miller-Jensen
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Nuclear Translocation ◽  
Single Cells ◽  
Live Cell ◽  
Relative Level ◽  
Fluorescent Reporter ◽  
Cytokines And Chemokines ◽  
Cell Processes ◽  
Cell To Cell Variability

Abstract Innate immune cells, including macrophages and dendritic cells, protect the host from pathogenic assaults in part through secretion of a program of cytokines and chemokines (C/Cs). Cell-to-cell variability in C/C secretion appears to contribute to the regulation of the immune response, but the sources of secretion variability are largely unknown. To begin to track the biological sources that control secretion variability, we developed and validated a microfluidic device to integrate live-cell imaging of fluorescent reporter proteins with a single-cell assay of protein secretion. We used this device to image NF-κB RelA nuclear translocation dynamics and Tnf transcription dynamics in macrophages in response to stimulation with the bacterial component lipopolysaccharide (LPS), followed by quantification of secretion of TNF, CCL2, CCL3, and CCL5. We found that the timing of the initial peak of RelA signaling in part determined the relative level of TNF and CCL3 secretion, but not CCL2 and CCL5 secretion. Our results support evidence that differences in timing across cell processes partly account for cell-to-cell variability in downstream responses, but that other factors introduce variability at each biological step.

scholarly journals Towards best-practice approaches for CRISPR/Cas9 gene engineering

2018 ◽  
Author(s):  
Claude Van Campenhout ◽  
Pauline Cabochette ◽  
Anne-Clémence Veillard ◽  
Miklos Laczik ◽  
Agnieszka Zelisko-Schmidt ◽  
...  
Keyword(s):  
Genome Editing ◽  
Best Practice ◽  
Rapid Diagnostics ◽  
Gene Engineering ◽  
Guide Rna ◽  
Technical Parameters ◽  
A Genome ◽  
Transcriptional Modulation ◽  
Fundamental Research ◽  
Research Drug

AbstractIn recent years, CRISPR has evolved from “the curious sequence of unknown biological function” into a functional genome editing tool. The CRISPR/Cas9 technology is now delivering novel genetic models for fundamental research, drug screening, therapy development, rapid diagnostics and transcriptional modulation. Despite the apparent simplicity of the CRISPR/Cas9 system, the outcome of a genome editing experiment can be substantially impacted by technical parameters as well as biological considerations. Here, we present guidelines and tools to optimize CRISPR/Cas9 genome targeting efficiency and specificity. The nature of the target locus, the design of the single guide RNA and the choice of the delivery method should all be carefully considered prior to a genome editing experiment. Different methods can also be used to detect off-target cleavages and decrease the risk of unwanted mutations. Together, these optimized tools and proper controls are essential to the assessment of CRISPR/Cas9 genome editing experiments.

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