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

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.

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Regulated processive transcription of chromatin by T7 RNA polymerase in Trypanosoma brucei

Nucleic Acids Research ◽

10.1093/nar/26.20.4626 ◽

1998 ◽

Vol 26 (20) ◽

pp. 4626-4634 ◽

Cited By ~ 81

Author(s):

E. Wirtz ◽

M. Hoek ◽

G. A. M. Cross

Keyword(s):

Rna Polymerase ◽

Trypanosoma Brucei ◽

T7 Rna Polymerase

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A modular and optimized single marker system for generating Trypanosoma brucei cell lines expressing T7 RNA polymerase and the tetracycline repressor

Open Biology ◽

10.1098/rsob.110037 ◽

2012 ◽

Vol 2 (2) ◽

pp. 110037 ◽

Cited By ~ 61

Author(s):

S. K. Poon ◽

L. Peacock ◽

W. Gibson ◽

K. Gull ◽

S. Kelly

Keyword(s):

Rna Polymerase ◽

Trypanosoma Brucei ◽

Cell Lines ◽

Genome Organization ◽

Tsetse Fly ◽

T7 Rna Polymerase ◽

Vector System ◽

Single Marker ◽

Tetracycline Repressor

Here, we present a simple modular extendable vector system for introducing the T7 RNA polymerase and tetracycline repressor genes into Trypanosoma brucei . This novel system exploits developments in our understanding of gene expression and genome organization to produce a streamlined plasmid optimized for high levels of expression of the introduced transgenes. We demonstrate the utility of this novel system in bloodstream and procyclic forms of Trypanosoma brucei , including the genome strain TREU927/4. We validate these cell lines using a variety of inducible experiments that recapture previously published lethal and non-lethal phenotypes. We further demonstrate the utility of the single marker (SmOx) TREU927/4 cell line for in vivo experiments in the tsetse fly and provide a set of plasmids that enable both whole-fly and salivary gland-specific inducible expression of transgenes.

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A New Generation of T7 RNA Polymerase-Independent Inducible Expression Plasmids for Trypanosoma brucei

PLoS ONE ◽

10.1371/journal.pone.0035167 ◽

2012 ◽

Vol 7 (4) ◽

pp. e35167 ◽

Cited By ~ 16

Author(s):

Jack Sunter ◽

Bill Wickstead ◽

Keith Gull ◽

Mark Carrington

Keyword(s):

Rna Polymerase ◽

Trypanosoma Brucei ◽

Inducible Expression ◽

T7 Rna Polymerase ◽

New Generation ◽

Expression Plasmids

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

10.21203/rs.3.rs-21224/v1 ◽

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.

Download Full-text