scholarly journals High-fidelity Cas13 variants for targeted RNA degradation with minimal collateral effect

2021 ◽  
Author(s):  
Huawei Tong ◽  
Jia Huang ◽  
Qingquan Xiao ◽  
Bingbing He ◽  
Xue Dong ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Basic Research ◽  
Rna Degradation ◽  
High Fidelity ◽  
Reporter System ◽  
Fluorescent Reporter ◽  
Major Barrier ◽  
Collateral Effects ◽  
Collateral Effect

CRISPR-Cas13 systems have recently been employed for targeted RNA degradation in various organisms. However, collateral degradation of bystander RNAs has imposed a major barrier for their in vivo applications. We designed a dual-fluorescent reporter system for detecting collateral effects and screening Cas13 variants in mammalian cells. Among over 200 engineered variants, several Cas13 variants (including Cas13d and Cas13X) exhibit efficient on-target activity but markedly reduced collateral activity. Furthermore, transcriptome-wide off-targets and cell growth arrest induced by Cas13 are absent for these variants. Importantly, high-fidelity Cas13 variants show comparable RNA knockdown activity with wild-type Cas13 but no detectable collateral damage in transgenic mice and adeno-associated virus-mediated somatic cell targeting. Thus, high-fidelity Cas13 variants with minimal collateral effect are now available for targeted degradation of RNAs in basic research and therapeutic applications.

scholarly journals Genetically encoded betaxanthin-based small-molecular fluorescent reporter for mammalian cells

2020 ◽  
Vol 48 (12) ◽  
pp. e67-e67
Author(s):  
Pascal Stücheli ◽  
Simon Sieber ◽  
David W Fuchs ◽  
Leo Scheller ◽  
Tobias Strittmatter ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Human Model ◽  
Fluorescence Labeling ◽  
Application Software ◽  
Reporter System ◽  
Fluorescent Reporter ◽  
Amanita Muscaria ◽  
Standard Application ◽  
Fungal Pigments ◽  
Heterologous Pathway

Abstract We designed and engineered a dye production cassette encoding a heterologous pathway, including human tyrosine hydroxylase and Amanita muscaria 4,5-DOPA dioxygenase, for the biosynthesis of the betaxanthin family of plant and fungal pigments in mammalian cells. The system does not impair cell viability, and can be used as a non-protein reporter system to directly visualize the dynamics of gene expression by profiling absorbance or fluorescence in the supernatant of cell cultures, as well as for fluorescence labeling of individual cells. Pigment profiling can also be multiplexed with reporter proteins such as mCherry or the human model glycoprotein SEAP (secreted alkaline phosphatase). Furthermore, absorbance measurement with a smartphone camera using standard application software enables inexpensive, low-tech reporter quantification.

scholarly journals Red Light/Green Light, a Dual Fluorescent Protein Reporter System To Study Enhancer-Promoter Specificity in Drosophila

2020 ◽  
Vol 10 (3) ◽  
pp. 985-997
Author(s):  
Eric M. Camino ◽  
Micheal L. Weinstein ◽  
Mary P. List ◽  
Jordan E. Vellky ◽  
Mark Rebeiz ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Red Light ◽  
Control Element ◽  
Gene Promoters ◽  
Spatial And Temporal Patterns ◽  
Reporter System ◽  
Fluorescent Reporter ◽  
Promoter Specificity ◽  
Reporter Transgene

Enhancers activate gene transcription in spatial and temporal patterns by interactions with gene promoters. These elements typically reside distal to their target promoter, with which they must interact selectively. Additional elements may contribute to enhancer-promoter specificity, including remote control element sequences within enhancers, tethering elements near promoters, and insulator/boundary elements that disrupt off-target interactions. However, few of these elements have been mapped, and as a result, the mechanisms by which these elements interact remain poorly understood. One impediment is their method of study, namely reporter transgenes in which enhancers are placed adjacent to a heterologous promoter, which may circumvent mechanisms controlling enhancer-promoter specificity and long-range interactions. Here, we report an optimized dual reporter transgene system in Drosophila melanogaster that allows the simultaneous comparison of an enhancer’s ability to activate proximal and distal fluorescent reporter genes. Testing a panel of fluorescent transgenes in vivo, we found a two-protein combination that allows simultaneous measurement with minimal detection interference. We note differences among four tested enhancers in their ability to regulate a distally placed reporter transgene. These results suggest that enhancers differ in their requirements for promoter interaction and raise important practical considerations when studying enhancer function.

scholarly journals Visualizing homologous recombination and illustrating DNA repair pathway interaction in vivo via a bioengineered fluorescent reporter system

2012 ◽  
Vol 26 (S1) ◽  
Author(s):  
Michelle R. Sukup Jackson ◽  
Orsolya Kiraly ◽  
Elizabeth Rowaland ◽  
Kelly Schulte ◽  
Laura J Niedernhofer ◽  
...  
Keyword(s):  
Dna Repair ◽  
Homologous Recombination ◽  
Repair Pathway ◽  
Reporter System ◽  
Fluorescent Reporter ◽  
Pathway Interaction

scholarly journals In Vivo Kinetics of mRNA Splicing and Transport in Mammalian Cells

2002 ◽  
Vol 22 (19) ◽  
pp. 6706-6718 ◽  
Author(s):  
A. Audibert ◽  
D. Weil ◽  
F. Dautry
Keyword(s):  
Mammalian Cells ◽  
Rna Degradation ◽  
Complete Analysis ◽  
First Order ◽  
Cellular Mrnas ◽  
Kinetics Of ◽  
Progressive Accumulation ◽  
Transcriptional Induction

ABSTRACT The kinetics of pre-mRNA processing in living cells is poorly known, preventing a detailed analysis of the regulation of these reactions. Using tetracycline-regulated promoters we performed, during a transcriptional induction, a complete analysis of the maturation of two cellular mRNAs, those for LT-α and β-globin. In both cases, splicing was appropriately described by first-order reactions with corresponding half-lives ranging between 0.4 and 7.5 min, depending on the intron. Transport also behaved as a first-order reaction during the early phase of β-globin expression, with a nuclear dwelling time of 4 min. At a later time, analysis was prevented by the progressive accumulation within the nucleus of mature mRNA not directly involved in export. Our results further establish for these genes that (i) splicing components are never limiting, even when expression is induced in naive cells, (ii) there is no significant RNA degradation during splicing and transport, and (iii) precursor-to-product ratios at steady state can be used for the determination of splicing rates. Finally, the comparison between the kinetics of splicing during transcriptional induction and during transcriptional shutoff reveals a novel coupling between transcription and splicing.

scholarly journals Initiator-Elongator Discrimination in Vertebrate tRNAs for Protein Synthesis

1998 ◽  
Vol 18 (3) ◽  
pp. 1459-1466 ◽  
Author(s):  
Harold J. Drabkin ◽  
Melanie Estrella ◽  
Uttam L. Rajbhandary
Keyword(s):  
Protein Synthesis ◽  
Mammalian Cells ◽  
Elongation Factor ◽  
Reporter System ◽  
Base Pairs ◽  
Initiator Trna ◽  
Phosphate Backbone ◽  
Elongation Step

ABSTRACT Initiator tRNAs are used exclusively for initiation of protein synthesis and not for the elongation step. We show, in vivo and in vitro, that the primary sequence feature that prevents the human initiator tRNA from acting in the elongation step is the nature of base pairs 50:64 and 51:63 in the TΨC stem of the initiator tRNA. Various considerations suggest that this is due to sequence-dependent perturbation of the sugar phosphate backbone in the TΨC stem of initiator tRNA, which most likely blocks binding of the elongation factor to the tRNA. Because the sequences of all vertebrate initiator tRNAs are identical, our findings with the human initiator tRNA are likely to be valid for all vertebrate systems. We have developed reporter systems that can be used to monitor, in mammalian cells, the activity in elongation of mutant human initiator tRNAs carrying anticodon sequence mutations from CAU to CCU (the C35 mutant) or to CUA (the U35A36 mutant). Combination of the anticodon sequence mutation with mutations in base pairs 50:64 and 51:63 yielded tRNAs that act as elongators in mammalian cells. Further mutation of the A1:U72 base pair, which is conserved in virtually all eukaryotic initiator tRNAs, to G1:C72 in the C35 mutant background yielded tRNAs that were even more active in elongation. In addition, in a rabbit reticulocyte in vitro protein-synthesizing system, a tRNA carrying the TΨC stem and the A1:U72-to-G1:C72 mutations was almost as active in elongation as the elongator methionine tRNA. The combination of mutant initiator tRNA with the CCU anticodon and the reporter system developed here provides the first example of missense suppression in mammalian cells.

scholarly journals An Improved Recombination-BasedIn VivoExpression Technology-Like Reporter System Reveals DifferentialcyaAGene Activation in Bordetella Species

2013 ◽  
Vol 81 (4) ◽  
pp. 1295-1305 ◽  
Author(s):  
Matthew S. Byrd ◽  
Eliza Mason ◽  
Michael W. Henderson ◽  
Erich V. Scheller ◽  
Peggy A. Cotter
Keyword(s):  
Virulence Factors ◽  
Gene Activation ◽  
Regulatory System ◽  
Reporter System ◽  
Fluorescent Reporter ◽  
Content Type ◽  
Genes Encoding ◽  
Species Specific

ABSTRACTBordetella pertussisandBordetella bronchisepticarely on the global two-component regulatory system BvgAS to control expression of distinct phenotypic phases. In the Bvg−phase, expression ofvrggenes, including those required for motility inB. bronchiseptica, is activated and genes encoding virulence factors are not expressed. Conversely, in the Bvg+phase, genes encoding virulence factors are highly expressed while genes necessary for motility are repressed. Although several genetic analyses have demonstrated the importance of the Bvg+phase during respiratory infection, Bvg-regulated gene activation inB. bronchisepticahas not been investigatedin vivo. To address this, we developed a plasmid, pGFLIP, that encodes a sensitive Flp recombinase-based fluorescent reporter system able to document gene activation bothin vitroandin vivo. Using pGFLIP, we demonstrated thatcyaA, considered to be a “late” Bvg+phase gene, is activated substantially earlier inB. bronchisepticathanB. pertussisfollowing a switch from Bvg−to Bvg+phase conditions. We show that the altered activation ofcyaAis not due to differences in thecyaApromoter or in thebvgASalleles ofB. bronchisepticacompared toB. pertussis, but appears to be species specific. Finally, we used pGFLIP to show thatflaAremains repressed during infection, confirming thatB. bronchisepticadoes not modulate to the Bvg−phasein vivo.

scholarly journals Multi-Stress Induction of the Mycobacterium tuberculosis MbcTA Bactericidal Toxin-Antitoxin System

Toxins ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 329
Author(s):  
Kanchiyaphat Ariyachaokun ◽  
Anna D. Grabowska ◽  
Claude Gutierrez ◽  
Olivier Neyrolles
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Internal Standard ◽  
Physical Interaction ◽  
Genetic Dissection ◽  
Reporter System ◽  
Fluorescent Reporter ◽  
Promising Target

MbcTA is a type II toxin/antitoxin (TA) system of Mycobacterium tuberculosis. The MbcT toxin triggers mycobacterial cell death in vitro and in vivo through the phosphorolysis of the essential metabolite NAD+ and its bactericidal activity is neutralized by physical interaction with its cognate antitoxin MbcA. Therefore, the MbcTA system appears as a promising target for the development of novel therapies against tuberculosis, through the identification of compounds able to antagonize or destabilize the MbcA antitoxin. Here, the expression of the mbcAT operon and its regulation were investigated. A dual fluorescent reporter system was developed, based on an integrative mycobacterial plasmid that encodes a constitutively expressed reporter, serving as an internal standard for monitoring mycobacterial gene expression, and an additional reporter, dependent on the promoter under investigation. This system was used both in M. tuberculosis and in the fast growing model species Mycobacterium smegmatis to: (i) assess the autoregulation of mbcAT; (ii) perform a genetic dissection of the mbcA promoter/operator region; and (iii) explore the regulation of mbcAT transcription from the mbcA promoter (PmbcA) in a variety of stress conditions, including in vivo in mice and in macrophages.

scholarly journals Regulation of Ribosomal Protein Synthesis in Mycobacteria: The Autogenous Control of rpsO

2021 ◽  
Vol 22 (18) ◽  
pp. 9679
Author(s):  
Leonid V. Aseev ◽  
Ludmila S. Koledinskaya ◽  
Oksana S. Bychenko ◽  
Irina V. Boni
Keyword(s):  
Protein Synthesis ◽  
Ribosomal Protein ◽  
Shuttle Vector ◽  
Ectopic Expression ◽  
Egfp Expression ◽  
Reporter System ◽  
Fluorescent Reporter ◽  
E Coli ◽  
Ribosomal Protein Synthesis

The autogenous regulation of ribosomal protein (r-protein) synthesis plays a key role in maintaining the stoichiometry of ribosomal components in bacteria. In this work, taking the rpsO gene as a classic example, we addressed for the first time the in vivo regulation of r-protein synthesis in the mycobacteria M. smegmatis (Msm) and M. tuberculosis (Mtb). We used a strategy based on chromosomally integrated reporters under the control of the rpsO regulatory regions and the ectopic expression of Msm S15 to measure its impact on the reporter expression. Because the use of E. coli as a host appeared inefficient, a fluorescent reporter system was developed by inserting Msm or Mtb rpsO-egfp fusions into the Msm chromosome and expressing Msm S15 or E. coli S15 in trans from a novel replicative shuttle vector, pAMYC. The results of the eGFP expression measurements in Msm cells provided evidence that the rpsO gene in Msm and Mtb was feedback-regulated at the translation level. The mutagenic analysis showed that the folding of Msm rpsO 5′UTR in a pseudoknot appeared crucial for repression by both Msm S15 and E. coli S15, thus indicating a striking resemblance of the rpsO feedback control in mycobacteria and in E. coli.

scholarly journals Modelling genetic mosaicism of neurodevelopmental disorders in vivo by a Cre-amplifying fluorescent reporter

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Francesco Trovato ◽  
Riccardo Parra ◽  
Enrico Pracucci ◽  
Silvia Landi ◽  
Olga Cozzolino ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Reporter System ◽  
Fluorescent Reporter ◽  
Neuronal Phenotype ◽  
Two Photon Microscopy ◽  
Two Photon ◽  
Genetic Mosaicism ◽  
Monogenic Diseases ◽  
The Central Nervous System

AbstractGenetic mosaicism, a condition in which an organ includes cells with different genotypes, is frequently present in monogenic diseases of the central nervous system caused by the random inactivation of the X-chromosome, in the case of X-linked pathologies, or by somatic mutations affecting a subset of neurons. The comprehension of the mechanisms of these diseases and of the cell-autonomous effects of specific mutations requires the generation of sparse mosaic models, in which the genotype of each neuron is univocally identified by the expression of a fluorescent protein in vivo. Here, we show a dual-color reporter system that, when expressed in a floxed mouse line for a target gene, leads to the creation of mosaics with tunable degree. We demonstrate the generation of a knockout mosaic of the autism/epilepsy related gene PTEN in which the genotype of each neuron is reliably identified, and the neuronal phenotype is accurately characterized by two-photon microscopy.

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