scholarly journals A transient CRISPR/Cas9 expression system for genome editing in Trypanosoma brucei

2020 ◽  
Author(s):  
Sebastian Shaw ◽  
Sebastian Knüsel ◽  
Sarah Hoenner ◽  
Isabel Roditi
Keyword(s):  
Rna Polymerase ◽  
Cell Line ◽  
Genome Editing ◽  
Trypanosoma Brucei ◽  
Transient Expression ◽  
Expression System ◽  
T7 Rna Polymerase ◽  
A Genome ◽  
Pcr Products

Abstract Objective Generation of knockouts and in situ tagging of genes in Trypanosoma brucei has been greatly facilitated by using CRISPR/Cas9 as a genome editing tool. To date, this has entailed using a limited number of cell lines that are stably transformed to express Cas9 and T7 RNA polymerase (T7RNAP). It would be desirable, however, to be able to use CRISPR/Cas9 for any trypanosome cell line. Results We describe a sequential transfection expression system that enables transient expression of the two proteins, followed by delivery of PCR products for gRNAs and repair templates. This procedure can be used for genome editing without the need for stable integration of the Cas9 and T7RNAP genes.

scholarly journals A transient CRISPR/Cas9 expression system for genome editing in Trypanosoma brucei

2020 ◽  
Author(s):  
Sebastian Shaw ◽  
Sebastian Knüsel ◽  
Sarah Hoenner ◽  
Isabel Roditi
Keyword(s):  
Rna Polymerase ◽  
Genome Editing ◽  
Trypanosoma Brucei ◽  
Transient Expression ◽  
Cell Lines ◽  
Expression System ◽  
T7 Rna Polymerase ◽  
A Genome ◽  
Pcr Products

Abstract ObjectiveGeneration of knockouts and in situ tagging of genes in Trypanosoma brucei has been greatly facilitated by using CRISPR/Cas9 as a genome editing tool. To date, this has entailed using a limited number of cell lines that are stably transformed to express Cas9 and T7 RNA polymerase (T7RNAP). It would be desirable, however, to be able to use CRISPR/Cas9 for any cell line.ResultsWe describe a sequential transfection expression system that enables transient expression of the two proteins, followed by delivery of PCR products for gRNAs and repair templates. This procedure can be used for genome editing without the need for stable integration of the Cas9 and T7RNAP genes.

scholarly journals Improved In Situ Hybridization to HIV with RNA Probes Derived from PCR Products

1997 ◽  
Vol 45 (5) ◽  
pp. 721-727 ◽  
Author(s):  
Richard W. Cone ◽  
Erika Schlaepfer
Keyword(s):  
In Situ Hybridization ◽  
Rna Polymerase ◽  
Plasmid Dna ◽  
T7 Rna Polymerase ◽  
Rna Probes ◽  
Pcr Products ◽  
Rna Polymerase Promoter ◽  
Hiv 1 ◽  
Specific Mrna

These experiments tested the hypothesis that a pool of PCR-derived RNA probes with defined length and even representation of the target sequences could produce more specific and intense in situ hybridization signals than randomly size-reduced, plasmid-derived RNA probes. In situ hybridization was performed with sense and anti-sense HIV-1 RNA probes that were derived from PCR products tailed with the T7 RNA polymerase promoter or from plasmid DNA. In situ hybridization using a pool of seven anti-sense or sense PCR-derived RNA probes (1805 nucleotides of HIV sequence, 257 nucleotides average probe length) was compared with hybridization using anti-sense or sense RNA probes made from a plasmid representing the HIV-1 env gene (3151 nucleotides of HIV-1 target). The pooled PCR-derived probes resulted in stronger in situ hybridization signals and less background than those produced with plasmid-derived RNA probes. This method for creating PCR-derived RNA probes improves the feasibility of synthesizing multiple, discrete RNA probes for studies of specific mRNA expression because it does not require the subcloning steps used to construct plasmids. PCR-derived RNA probes may provide a viable alternative to the use of plasmid-derived RNA probes for in situ hybridization.

scholarly journals A Reverse Genetics System for Cypovirus Based on a Bacmid Expressing T7 RNA Polymerase

Viruses ◽  
2019 ◽  
Vol 11 (4) ◽  
pp. 314 ◽  
Author(s):  
Gaobo Zhang ◽  
Jian Yang ◽  
Fujun Qin ◽  
Congrui Xu ◽  
Jia Wang ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Reverse Genetics ◽  
T7 Rna Polymerase ◽  
Sf9 Cells ◽  
Genomic Segment ◽  
Egfp Gene ◽  
A Genome ◽  
Typical Morphology ◽  
Occlusion Bodies ◽  
Rescue System

Dendrolimus punctatus cypovirus (DpCPV), belonging to the genus Cypovirus within the family Reoviridae, is considered the most destructive pest of pine forests worldwide. DpCPV has a genome consisting of 10 linear double-stranded RNA segments. To establish a reverse genetics system, we cloned cDNAs encoding the 10 genomic segments of DpCPV into three reverse genetics vectors in which each segment was transcribed under the control of a T7 RNA polymerase promoter and terminator tagged with a hepatitis delta virus ribozyme sequence. We also constructed a vp80-knockout Autographa californica multiple nucleopolyhedrovirus bacmid to express a T7 RNA polymerase codon-optimized for Sf9 cells. Following transfection of Sf9 cells with the three vectors and the bacmid, occlusion bodies (OBs) with the typical morphology of cypovirus polyhedra were observed by optical microscopy. The rescue system was verified by incorporation of a HindIII restriction enzyme site null mutant of the 9th genomic segment. Furthermore, when we co-transfected Sf9 cells with the reverse genetics vectors, the bacmid, and an additional vector bearing an egfp gene flanked with the 5′ and 3′ untranslated regions of the 10th genomic segment, aggregated green fluorescence co-localizing with the OBs was observed. The rescued OBs were able to infect Spodopetra exigua larvae, although their infectivity was significantly lower than that of wild-type DpCPV. This reverse genetics system for DpCPV could be used to explore viral replication and pathogenesis and to facilitate the development of novel bio-insecticides and expression systems for exogenous proteins.

High-level transcription of large gene regions: a novel T7 RNA-polymerase-based system for expression of functional hydrogenases in the phototrophic bacterium Rhodobacter capsulatus

2005 ◽  
Vol 33 (1) ◽  
pp. 56-58 ◽  
Author(s):  
T. Drepper ◽  
S. Arvani ◽  
F. Rosenau ◽  
S. Wilhelm ◽  
K.-E. Jaeger
Keyword(s):  
Rna Polymerase ◽  
Rhodobacter Capsulatus ◽  
Expression System ◽  
Active Form ◽  
T7 Rna Polymerase ◽  
High Level Synthesis ◽  
Phototrophic Bacterium ◽  
Large Gene ◽  
High Level ◽  
Complex Enzymes

High-level synthesis of complex enzymes like bacterial [NiFe] hydrogenases, in general, requires an expression system that allows concerted expression of a large number of genes. So far, it has not been possible to overproduce a hydrogenase in a stable and active form by using a customary expression system. Therefore we started to establish a new, T7-based expression system in the phototrophic bacterium Rhodobacter capsulatus. The beneficial properties of this bacterial host in combination with the unique capacity of T7 RNA polymerase to synthesize long transcripts will allow the high-level synthesis and assembly of active hydrogenase as well as other complex enzymes in the near future.

T7 RNA polymerase-driven transcription in mitochondria of Leishmania tarentolae and Trypanosoma brucei

1999 ◽  
Vol 103 (2) ◽  
pp. 251-259 ◽  
Author(s):  
Antonio M Estévez ◽  
Otavio H Thiemann ◽  
Juan D Alfonzo ◽  
Larry Simpson
Keyword(s):  
Rna Polymerase ◽  
Trypanosoma Brucei ◽  
T7 Rna Polymerase ◽  
Leishmania Tarentolae

scholarly journals Engineering efficient termination of bacteriophage T7 RNA polymerase transcription

2021 ◽  
Author(s):  
Diana Gabriela Calvopina Chavez ◽  
Mikaela Anne Gardner ◽  
Joel S Griffitts
Keyword(s):  
Rna Polymerase ◽  
Molecular Level ◽  
Expression System ◽  
T7 Rna Polymerase ◽  
Bacteriophage T7 ◽  
Lower Efficiency ◽  
Transcriptional Termination ◽  
T7 Polymerase

The bacteriophage T7 expression system is one of the most prominent transcription systems used in biotechnology and molecular-level research. However, T7 RNA polymerase is prone to read-through transcription due to its high processivity. As a consequence, enforcing efficient transcriptional termination is difficult. The termination hairpin found natively in the T7 genome is adapted to be inefficient, exhibiting 62% termination efficiency in vivo and even lower efficiency in vitro. In this study, we engineered a series of sequences that outperform the efficiency of the native terminator hairpin. By embedding a previously discovered 8-nucleotide T7 polymerase pause sequence within a synthetic hairpin sequence, we observed in vivo termination efficiency of 91%; by joining two short sequences into a tandem 2-hairpin structure, termination efficiency was increased to 98% in vivo and 91% in vitro. This study also tests the ability of these engineered sequences to terminate transcription of the Escherichia coli RNA polymerase. Two out of three of the most successful T7 polymerase terminators also facilitated termination of the bacterial polymerase with around 99% efficiency.

scholarly journals Synthetic logic circuits using RNA aptamer against T7 RNA polymerase

2014 ◽  
Author(s):  
Jongmin Kim ◽  
Juan F Quijano ◽  
Enoch Yeung ◽  
Richard M Murray
Keyword(s):  
Rna Polymerase ◽  
Expression System ◽  
Building Blocks ◽  
Logic Circuits ◽  
T7 Rna Polymerase ◽  
Free Expression ◽  
Rna Aptamer ◽  
Cell Free Expression System

Recent advances in nucleic acids engineering introduced several RNA-based regulatory components for synthetic gene circuits, expanding the toolsets to engineer organisms. In this work, we designed genetic circuits implementing an RNA aptamer previously described to have the capability of binding to the T7 RNA polymerase and inhibiting its activity in vitro. Using in vitro transcription assays, we first demonstrated the utility of the RNA aptamer in combination with programmable synthetic transcription networks. As a step to quickly assess the feasibility of aptamer functions in vivo, a cell-free expression system was used as a breadboard to emulate the in vivo conditions of E. coli. We tested the aptamer and its three sequence variants in the cell-free expression system, verifying the aptamer functionality in the cell-free testbed. In vivo expression of aptamer and its variants demonstrated control over GFP expression driven by T7 RNA polymerase with different response curves, indicating its ability to serve as building blocks for both logic circuits and transcriptional cascades. This work elucidates the potential of RNA-based regulators for cell programming with improved controllability leveraging the fast production and degradation time scales of RNA molecules.

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