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Site-specific DNA double-strand breaks greatly increase stable transformation efficiency in Trypanosoma brucei.

Glover L, Horn D - Mol. Biochem. Parasitol. (2009)

Bottom Line: We have explored the effect of DSBs on targeted integration in bloodstream-form Trypanosoma brucei, focusing on the ribosomal RNA-spacer target commonly used to integrate recombinant constructs.DSB-repair within the ribosomal RNA tandem gene-repeats is likely dominated by single-strand annealing allowing approximately 80% of cells to survive the break.In the example presented, more than 1% of cells that survive the procedure were transformed generating 80,000 transformants from a typical experiment.

View Article: PubMed Central - PubMed

Affiliation: London School of Hygiene & Tropical Medicine, Keppel Street, London, WC1E 7HT, UK.

ABSTRACT
Genetic manipulation in African trypanosomes typically relies upon electroporation with chromosomal integration of DNA constructs by homologous recombination. Relatively little is known about chromosomal recombination and repair in these organisms however and low transformation efficiency and position effects can limit forward genetic approaches. In yeast and mammalian cells, site-specific DNA double-strand breaks (DSBs) stimulate targeted integration through homologous recombination-based repair where the exogenous DNA serves as the template. We have explored the effect of DSBs on targeted integration in bloodstream-form Trypanosoma brucei, focusing on the ribosomal RNA-spacer target commonly used to integrate recombinant constructs. DSB-repair within the ribosomal RNA tandem gene-repeats is likely dominated by single-strand annealing allowing approximately 80% of cells to survive the break. In the presence of exogenous DNA, transformation efficiency is increased approximately 250-fold by DSB-induction. In the example presented, more than 1% of cells that survive the procedure were transformed generating 80,000 transformants from a typical experiment.

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Related in: MedlinePlus

Response to a DSB at the RRNA-spacer locus. (A) The schematic map illustrates an RFP–PAC fusion gene (RsP) with an embedded I-SceI site (indicated by DSB) at the RRNA-spacer (RRNAs) locus. pRsPRRNA was assembled as follows: an RRNA promoter (PRRNA) fragment was amplified from genomic DNA using primers RpF (GATCcggcggTAGCTTTCCACCCAGCGC) and RpR (GATCcggccgggcccACTGggatccTCTGAGAGCGGTCAGTTGC), digested with EagI (relevant restriction sites in lower-case) and ligated to a NotI-digested RRNA-spacer fragment in pBlusescript. An RsP cassette was then added using the BspI201 and BamHI sites. The RsPRRNA construct was then digested with SacI/AgeI and introduced into the 2T1 bloodstream-form T. brucei strain [10] that also contained a tetracycline-inducible I-SceI ORF introduced using the pRPai construct [17]. These Lister 427, clone 221a cells were grown and manipulated as described [10]. (B) A clonogenic assay to assess recovery from a DSB. Cells in all un-induced wells tested remained puromycin-resistant and cells in every induced well were puromycin-sensitive indicating loss of the RsP cassette in the latter case. Cell counts were carried out using a haemocytometer and tetracycline (used at 1 μg ml−1) was from Sigma. Data are derived from a pair of independent RsPRRNA strains and error bars represent one standard deviation. (C) Physical monitoring of DNA resection adjacent to the lesion was carried out by slot-blot assay as described [1]. Genomic DNA samples were ‘native’, to detect ssDNA or denatured, to detect total DNA. The probes used on each blot are indicated on the right; the control probe is from chromosome 11 (Tb11.01.7240). Phoshorimager analysis was used to quantify the signals and ssRFP values were derived after correction for background, ssDNA versus total DNA and loading. The RFP ssDNA and total DNA plots indicate resection kinetics and DNA loss respectively.
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fig1: Response to a DSB at the RRNA-spacer locus. (A) The schematic map illustrates an RFP–PAC fusion gene (RsP) with an embedded I-SceI site (indicated by DSB) at the RRNA-spacer (RRNAs) locus. pRsPRRNA was assembled as follows: an RRNA promoter (PRRNA) fragment was amplified from genomic DNA using primers RpF (GATCcggcggTAGCTTTCCACCCAGCGC) and RpR (GATCcggccgggcccACTGggatccTCTGAGAGCGGTCAGTTGC), digested with EagI (relevant restriction sites in lower-case) and ligated to a NotI-digested RRNA-spacer fragment in pBlusescript. An RsP cassette was then added using the BspI201 and BamHI sites. The RsPRRNA construct was then digested with SacI/AgeI and introduced into the 2T1 bloodstream-form T. brucei strain [10] that also contained a tetracycline-inducible I-SceI ORF introduced using the pRPai construct [17]. These Lister 427, clone 221a cells were grown and manipulated as described [10]. (B) A clonogenic assay to assess recovery from a DSB. Cells in all un-induced wells tested remained puromycin-resistant and cells in every induced well were puromycin-sensitive indicating loss of the RsP cassette in the latter case. Cell counts were carried out using a haemocytometer and tetracycline (used at 1 μg ml−1) was from Sigma. Data are derived from a pair of independent RsPRRNA strains and error bars represent one standard deviation. (C) Physical monitoring of DNA resection adjacent to the lesion was carried out by slot-blot assay as described [1]. Genomic DNA samples were ‘native’, to detect ssDNA or denatured, to detect total DNA. The probes used on each blot are indicated on the right; the control probe is from chromosome 11 (Tb11.01.7240). Phoshorimager analysis was used to quantify the signals and ssRFP values were derived after correction for background, ssDNA versus total DNA and loading. The RFP ssDNA and total DNA plots indicate resection kinetics and DNA loss respectively.

Mentions: For these studies, a Red Fluorescent Protein (RFP) – Puromycin N-ACetyltransferase (PAC) fusion gene with an embedded I-SceI site (RsP) and driven by an RRNA promoter, was assembled and targeted to RRNA-spacers in cells with a conditional I-SceI gene (Fig. 1A). RNA polymerase I transcription stimulates homologous recombination in T. brucei[15] and RRNA-spacer loci exert position effects on integrated promoters [10] so we screened several RsPRRNA clones for direct RFP fluorescence and selected one with the highest expression to ensure robust transcription at the target site. The vast majority (>99%) of these RsPRRNA cells revert to puromycin sensitivity when grown in tetracycline (data not shown) indicating efficient I-SceI cleavage and disruption or loss of the RsP cassette (see Fig. 1A). RsPRRNA cells were then tested for transformation efficiency by inducing I-SceI expression 3 h prior to electroporation. This generated 8 × 10−5 transformants, 200-fold more than control cells (Table 1, expt. 4) and similar to the efficiency obtained when these conditions were used to target loci on chromosomes 1 and 11.


Site-specific DNA double-strand breaks greatly increase stable transformation efficiency in Trypanosoma brucei.

Glover L, Horn D - Mol. Biochem. Parasitol. (2009)

Response to a DSB at the RRNA-spacer locus. (A) The schematic map illustrates an RFP–PAC fusion gene (RsP) with an embedded I-SceI site (indicated by DSB) at the RRNA-spacer (RRNAs) locus. pRsPRRNA was assembled as follows: an RRNA promoter (PRRNA) fragment was amplified from genomic DNA using primers RpF (GATCcggcggTAGCTTTCCACCCAGCGC) and RpR (GATCcggccgggcccACTGggatccTCTGAGAGCGGTCAGTTGC), digested with EagI (relevant restriction sites in lower-case) and ligated to a NotI-digested RRNA-spacer fragment in pBlusescript. An RsP cassette was then added using the BspI201 and BamHI sites. The RsPRRNA construct was then digested with SacI/AgeI and introduced into the 2T1 bloodstream-form T. brucei strain [10] that also contained a tetracycline-inducible I-SceI ORF introduced using the pRPai construct [17]. These Lister 427, clone 221a cells were grown and manipulated as described [10]. (B) A clonogenic assay to assess recovery from a DSB. Cells in all un-induced wells tested remained puromycin-resistant and cells in every induced well were puromycin-sensitive indicating loss of the RsP cassette in the latter case. Cell counts were carried out using a haemocytometer and tetracycline (used at 1 μg ml−1) was from Sigma. Data are derived from a pair of independent RsPRRNA strains and error bars represent one standard deviation. (C) Physical monitoring of DNA resection adjacent to the lesion was carried out by slot-blot assay as described [1]. Genomic DNA samples were ‘native’, to detect ssDNA or denatured, to detect total DNA. The probes used on each blot are indicated on the right; the control probe is from chromosome 11 (Tb11.01.7240). Phoshorimager analysis was used to quantify the signals and ssRFP values were derived after correction for background, ssDNA versus total DNA and loading. The RFP ssDNA and total DNA plots indicate resection kinetics and DNA loss respectively.
© Copyright Policy
Related In: Results  -  Collection

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fig1: Response to a DSB at the RRNA-spacer locus. (A) The schematic map illustrates an RFP–PAC fusion gene (RsP) with an embedded I-SceI site (indicated by DSB) at the RRNA-spacer (RRNAs) locus. pRsPRRNA was assembled as follows: an RRNA promoter (PRRNA) fragment was amplified from genomic DNA using primers RpF (GATCcggcggTAGCTTTCCACCCAGCGC) and RpR (GATCcggccgggcccACTGggatccTCTGAGAGCGGTCAGTTGC), digested with EagI (relevant restriction sites in lower-case) and ligated to a NotI-digested RRNA-spacer fragment in pBlusescript. An RsP cassette was then added using the BspI201 and BamHI sites. The RsPRRNA construct was then digested with SacI/AgeI and introduced into the 2T1 bloodstream-form T. brucei strain [10] that also contained a tetracycline-inducible I-SceI ORF introduced using the pRPai construct [17]. These Lister 427, clone 221a cells were grown and manipulated as described [10]. (B) A clonogenic assay to assess recovery from a DSB. Cells in all un-induced wells tested remained puromycin-resistant and cells in every induced well were puromycin-sensitive indicating loss of the RsP cassette in the latter case. Cell counts were carried out using a haemocytometer and tetracycline (used at 1 μg ml−1) was from Sigma. Data are derived from a pair of independent RsPRRNA strains and error bars represent one standard deviation. (C) Physical monitoring of DNA resection adjacent to the lesion was carried out by slot-blot assay as described [1]. Genomic DNA samples were ‘native’, to detect ssDNA or denatured, to detect total DNA. The probes used on each blot are indicated on the right; the control probe is from chromosome 11 (Tb11.01.7240). Phoshorimager analysis was used to quantify the signals and ssRFP values were derived after correction for background, ssDNA versus total DNA and loading. The RFP ssDNA and total DNA plots indicate resection kinetics and DNA loss respectively.
Mentions: For these studies, a Red Fluorescent Protein (RFP) – Puromycin N-ACetyltransferase (PAC) fusion gene with an embedded I-SceI site (RsP) and driven by an RRNA promoter, was assembled and targeted to RRNA-spacers in cells with a conditional I-SceI gene (Fig. 1A). RNA polymerase I transcription stimulates homologous recombination in T. brucei[15] and RRNA-spacer loci exert position effects on integrated promoters [10] so we screened several RsPRRNA clones for direct RFP fluorescence and selected one with the highest expression to ensure robust transcription at the target site. The vast majority (>99%) of these RsPRRNA cells revert to puromycin sensitivity when grown in tetracycline (data not shown) indicating efficient I-SceI cleavage and disruption or loss of the RsP cassette (see Fig. 1A). RsPRRNA cells were then tested for transformation efficiency by inducing I-SceI expression 3 h prior to electroporation. This generated 8 × 10−5 transformants, 200-fold more than control cells (Table 1, expt. 4) and similar to the efficiency obtained when these conditions were used to target loci on chromosomes 1 and 11.

Bottom Line: We have explored the effect of DSBs on targeted integration in bloodstream-form Trypanosoma brucei, focusing on the ribosomal RNA-spacer target commonly used to integrate recombinant constructs.DSB-repair within the ribosomal RNA tandem gene-repeats is likely dominated by single-strand annealing allowing approximately 80% of cells to survive the break.In the example presented, more than 1% of cells that survive the procedure were transformed generating 80,000 transformants from a typical experiment.

View Article: PubMed Central - PubMed

Affiliation: London School of Hygiene & Tropical Medicine, Keppel Street, London, WC1E 7HT, UK.

ABSTRACT
Genetic manipulation in African trypanosomes typically relies upon electroporation with chromosomal integration of DNA constructs by homologous recombination. Relatively little is known about chromosomal recombination and repair in these organisms however and low transformation efficiency and position effects can limit forward genetic approaches. In yeast and mammalian cells, site-specific DNA double-strand breaks (DSBs) stimulate targeted integration through homologous recombination-based repair where the exogenous DNA serves as the template. We have explored the effect of DSBs on targeted integration in bloodstream-form Trypanosoma brucei, focusing on the ribosomal RNA-spacer target commonly used to integrate recombinant constructs. DSB-repair within the ribosomal RNA tandem gene-repeats is likely dominated by single-strand annealing allowing approximately 80% of cells to survive the break. In the presence of exogenous DNA, transformation efficiency is increased approximately 250-fold by DSB-induction. In the example presented, more than 1% of cells that survive the procedure were transformed generating 80,000 transformants from a typical experiment.

Show MeSH
Related in: MedlinePlus