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To nick or not to nick: comparison of I-SceI single- and double-strand break-induced recombination in yeast and human cells.

Katz SS, Gimble FS, Storici F - PLoS ONE (2014)

Bottom Line: We show that K223I I-SceI-driven recombination follows a different mechanism than wild-type I-SceI-driven recombination, thus indicating that the initial DNA break that stimulates recombination is not a low-level DSB but a nick.We also demonstrate that K223I I-SceI efficiently elevates gene targeting at loci distant from the break site in yeast cells.These findings establish the capability of the I-SceI nickase to enhance recombination in yeast and human cells, strengthening the notion that nicking enzymes could be effective tools in gene correction strategies for applications in molecular biology, biotechnology, and gene therapy.

View Article: PubMed Central - PubMed

Affiliation: School of Biology, Georgia Institute of Technology, Atlanta, Georgia, United States of America.

ABSTRACT
Genetic modification of a chromosomal locus to replace an existing dysfunctional allele with a corrected sequence can be accomplished through targeted gene correction using the cell's homologous recombination (HR) machinery. Gene targeting is stimulated by generation of a DNA double-strand break (DSB) at or near the site of correction, but repair of the break via non-homologous end-joining without using the homologous template can lead to deleterious genomic changes such as in/del mutations, or chromosomal rearrangements. By contrast, generation of a DNA single-strand break (SSB), or nick, can stimulate gene correction without the problems of DSB repair because the uncut DNA strand acts as a template to permit healing without alteration of genetic material. Here, we examine the ability of a nicking variant of the I-SceI endonuclease (K223I I-SceI) to stimulate gene targeting in yeast Saccharomyces cerevisiae and in human embryonic kidney (HEK-293) cells. K223I I-SceI is proficient in both yeast and human cells and promotes gene correction up to 12-fold. We show that K223I I-SceI-driven recombination follows a different mechanism than wild-type I-SceI-driven recombination, thus indicating that the initial DNA break that stimulates recombination is not a low-level DSB but a nick. We also demonstrate that K223I I-SceI efficiently elevates gene targeting at loci distant from the break site in yeast cells. These findings establish the capability of the I-SceI nickase to enhance recombination in yeast and human cells, strengthening the notion that nicking enzymes could be effective tools in gene correction strategies for applications in molecular biology, biotechnology, and gene therapy.

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A nick occurring in asynchronous or G1 arrested cells stimulates gene correction by oligonucleotides equally efficiently.Shown are frequency of Trp+ transformants by oligonucleotides when cells were asynchronous (left) or arrested in G1 (right) at the time of wild-type-I-SceI or K223I I-SceI breakage prior to oligonucleotide transformation and when the SSB is generated on the “Crick” strand. All data are presented as the median with range (n≥5). For the specific numerical values see Table S3E. Wild-type I-SceI strains used: SAS-227 and SAS-228. K223I I-SceI strains used: SAS-229 and SAS-230. D145A I-SceI strains used: SAS-231 and SAS-232.
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pone-0088840-g004: A nick occurring in asynchronous or G1 arrested cells stimulates gene correction by oligonucleotides equally efficiently.Shown are frequency of Trp+ transformants by oligonucleotides when cells were asynchronous (left) or arrested in G1 (right) at the time of wild-type-I-SceI or K223I I-SceI breakage prior to oligonucleotide transformation and when the SSB is generated on the “Crick” strand. All data are presented as the median with range (n≥5). For the specific numerical values see Table S3E. Wild-type I-SceI strains used: SAS-227 and SAS-228. K223I I-SceI strains used: SAS-229 and SAS-230. D145A I-SceI strains used: SAS-231 and SAS-232.

Mentions: To demonstrate that the K223I I-SceI nicking activity, rather than its low DSB activity, is responsible for the observed Rad51-dependent gene correction by oligonucleotides, we determined if gene correction stimulated by wild-type I-SceI was dependent on Rad51 under conditions of low I-SceI expression. We adjusted the galactose concentration (0.02%) in the media to express sufficient wild-type I-SceI to stimulate gene correction 10-fold (p = 0.0043) or 4.2-fold (p = 0.0022) by the F or R oligonucleotides, respectively, above the level obtained when D145A I-SceI is expressed (after subtraction of the background). This level of gene targeting stimulation is approximately equal to that observed using the F and R oligonucleotides when K223I I-SceI is expressed in RAD51 cells in media containing 2% galactose (Figure 3D, left, light blue bars relative to yellow bars after background subtraction are compared to orange bars relative to yellow bars presented in Figure 3B left). Even at these lower frequencies of gene correction by using the F and R oligonucleotides, deletion of RAD51 still stimulated recombination (3.6-fold increase for F, p = 0.0152, and 2.9-fold increase for R, p = 0.0411, respectively) with wild-type I-SceI as compared to D145A control (Figure 3D, right). We also note that the background of Trp+ cells obtained with the no-oligonucleotide control is substantial only when wild-type I-SceI is expressed, even in low galactose concentration, and the nucleotide sequence of TRP5 in these cells differs from the sequence when an oligonucleotide is used to repair the lesion (Figure S1), suggesting an end joining mechanism of DSB repair in the absence of a repair template. Differently, the background of Trp+ cells for the no-oligonucleotide control was always <1 per 107 viable cells following expression of K223I I-SceI (Figure 3B-C and see also Figure 4), indicating that the broken trp5 marker cannot be repaired by end joining of the broken ends. These data are in line with the notion that only the break caused by wild-type I-SceI and not the one caused by K223I I-SceI is a DSB, which can be repaired by NHEJ. Overall, these results are strongly consistent with direct SSB-stimulated gene correction rather than gene correction promoted by low DSB levels following expression of K223I I-SceI. These findings indicate that the K223I I-SceI break is different from the one generated by wild-type I-SceI, and we conclude that the initial gene correction-stimulating lesion is an SSB.


To nick or not to nick: comparison of I-SceI single- and double-strand break-induced recombination in yeast and human cells.

Katz SS, Gimble FS, Storici F - PLoS ONE (2014)

A nick occurring in asynchronous or G1 arrested cells stimulates gene correction by oligonucleotides equally efficiently.Shown are frequency of Trp+ transformants by oligonucleotides when cells were asynchronous (left) or arrested in G1 (right) at the time of wild-type-I-SceI or K223I I-SceI breakage prior to oligonucleotide transformation and when the SSB is generated on the “Crick” strand. All data are presented as the median with range (n≥5). For the specific numerical values see Table S3E. Wild-type I-SceI strains used: SAS-227 and SAS-228. K223I I-SceI strains used: SAS-229 and SAS-230. D145A I-SceI strains used: SAS-231 and SAS-232.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3928301&req=5

pone-0088840-g004: A nick occurring in asynchronous or G1 arrested cells stimulates gene correction by oligonucleotides equally efficiently.Shown are frequency of Trp+ transformants by oligonucleotides when cells were asynchronous (left) or arrested in G1 (right) at the time of wild-type-I-SceI or K223I I-SceI breakage prior to oligonucleotide transformation and when the SSB is generated on the “Crick” strand. All data are presented as the median with range (n≥5). For the specific numerical values see Table S3E. Wild-type I-SceI strains used: SAS-227 and SAS-228. K223I I-SceI strains used: SAS-229 and SAS-230. D145A I-SceI strains used: SAS-231 and SAS-232.
Mentions: To demonstrate that the K223I I-SceI nicking activity, rather than its low DSB activity, is responsible for the observed Rad51-dependent gene correction by oligonucleotides, we determined if gene correction stimulated by wild-type I-SceI was dependent on Rad51 under conditions of low I-SceI expression. We adjusted the galactose concentration (0.02%) in the media to express sufficient wild-type I-SceI to stimulate gene correction 10-fold (p = 0.0043) or 4.2-fold (p = 0.0022) by the F or R oligonucleotides, respectively, above the level obtained when D145A I-SceI is expressed (after subtraction of the background). This level of gene targeting stimulation is approximately equal to that observed using the F and R oligonucleotides when K223I I-SceI is expressed in RAD51 cells in media containing 2% galactose (Figure 3D, left, light blue bars relative to yellow bars after background subtraction are compared to orange bars relative to yellow bars presented in Figure 3B left). Even at these lower frequencies of gene correction by using the F and R oligonucleotides, deletion of RAD51 still stimulated recombination (3.6-fold increase for F, p = 0.0152, and 2.9-fold increase for R, p = 0.0411, respectively) with wild-type I-SceI as compared to D145A control (Figure 3D, right). We also note that the background of Trp+ cells obtained with the no-oligonucleotide control is substantial only when wild-type I-SceI is expressed, even in low galactose concentration, and the nucleotide sequence of TRP5 in these cells differs from the sequence when an oligonucleotide is used to repair the lesion (Figure S1), suggesting an end joining mechanism of DSB repair in the absence of a repair template. Differently, the background of Trp+ cells for the no-oligonucleotide control was always <1 per 107 viable cells following expression of K223I I-SceI (Figure 3B-C and see also Figure 4), indicating that the broken trp5 marker cannot be repaired by end joining of the broken ends. These data are in line with the notion that only the break caused by wild-type I-SceI and not the one caused by K223I I-SceI is a DSB, which can be repaired by NHEJ. Overall, these results are strongly consistent with direct SSB-stimulated gene correction rather than gene correction promoted by low DSB levels following expression of K223I I-SceI. These findings indicate that the K223I I-SceI break is different from the one generated by wild-type I-SceI, and we conclude that the initial gene correction-stimulating lesion is an SSB.

Bottom Line: We show that K223I I-SceI-driven recombination follows a different mechanism than wild-type I-SceI-driven recombination, thus indicating that the initial DNA break that stimulates recombination is not a low-level DSB but a nick.We also demonstrate that K223I I-SceI efficiently elevates gene targeting at loci distant from the break site in yeast cells.These findings establish the capability of the I-SceI nickase to enhance recombination in yeast and human cells, strengthening the notion that nicking enzymes could be effective tools in gene correction strategies for applications in molecular biology, biotechnology, and gene therapy.

View Article: PubMed Central - PubMed

Affiliation: School of Biology, Georgia Institute of Technology, Atlanta, Georgia, United States of America.

ABSTRACT
Genetic modification of a chromosomal locus to replace an existing dysfunctional allele with a corrected sequence can be accomplished through targeted gene correction using the cell's homologous recombination (HR) machinery. Gene targeting is stimulated by generation of a DNA double-strand break (DSB) at or near the site of correction, but repair of the break via non-homologous end-joining without using the homologous template can lead to deleterious genomic changes such as in/del mutations, or chromosomal rearrangements. By contrast, generation of a DNA single-strand break (SSB), or nick, can stimulate gene correction without the problems of DSB repair because the uncut DNA strand acts as a template to permit healing without alteration of genetic material. Here, we examine the ability of a nicking variant of the I-SceI endonuclease (K223I I-SceI) to stimulate gene targeting in yeast Saccharomyces cerevisiae and in human embryonic kidney (HEK-293) cells. K223I I-SceI is proficient in both yeast and human cells and promotes gene correction up to 12-fold. We show that K223I I-SceI-driven recombination follows a different mechanism than wild-type I-SceI-driven recombination, thus indicating that the initial DNA break that stimulates recombination is not a low-level DSB but a nick. We also demonstrate that K223I I-SceI efficiently elevates gene targeting at loci distant from the break site in yeast cells. These findings establish the capability of the I-SceI nickase to enhance recombination in yeast and human cells, strengthening the notion that nicking enzymes could be effective tools in gene correction strategies for applications in molecular biology, biotechnology, and gene therapy.

Show MeSH
Related in: MedlinePlus