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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 K223I I-SceI break stimulates gene correction by oligonucleotides in yeast.(A) Scheme showing disrupted yeast trp5 chromosomal locus containing the I-SceI recognition sequence (black box). The position of the SSB is indicated (“Nick”) for the “Crick” and “Watson” constructs. Dashed gray lines indicate the complementarity between the F oligonucleotide and the antisense strand of the targeted gene, and between the R oligonucleotide and the sense strand of the targeted gene. (B–D) Frequencies of Trp+ transformants following expression of wild-type I-SceI (dark blue bars labeled “DSB”), K223I (orange bars labeled “SSB”), or D145A (yellow bars labeled “Non-break”) using either of the single or the pair of oligonucleotides to repair the break. All data are presented as the median with range (n≥5). For the specific numerical values see Table S3B-D. (B) Gene correction frequencies by oligonucleotides when an SSB is generated on the “Crick” (left) or “Watson” (right) chromosomal strand. (C) Frequency of transformants in RAD51 (left) or rad51  mutant (right) strains when the SSB is generated on the “Crick” strand. (D) Frequency of transformants following expression of wild-type I-SceI in RAD51 (left) or rad51  mutant (right) strains with final galactose concentrations of 2% (dark blue bars labeled “DSB (2%)”) or 0.02% (light blue bars labeled “DSB (0.02%)”). Wild-type I-SceI strains used: SAS-227 and SAS-228 (“Crick” and RAD51), SAS-281 and SAS-282 (“Watson”), and SAS-235 and SAS-236 (rad51Δ). K223I I-SceI strains used: SAS-229 and SAS-230 (“Crick” and RAD51), SAS-283 and SAS-284 (“Watson”), and SAS-237 and SAS-238 (rad51Δ). D145A I-SceI strains used: SAS-231 and SAS-232 (“Crick” and RAD51), SAS-285 and SAS-286 (“Watson”), and SAS-239 and SAS-240 (rad51Δ).
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pone-0088840-g003: A K223I I-SceI break stimulates gene correction by oligonucleotides in yeast.(A) Scheme showing disrupted yeast trp5 chromosomal locus containing the I-SceI recognition sequence (black box). The position of the SSB is indicated (“Nick”) for the “Crick” and “Watson” constructs. Dashed gray lines indicate the complementarity between the F oligonucleotide and the antisense strand of the targeted gene, and between the R oligonucleotide and the sense strand of the targeted gene. (B–D) Frequencies of Trp+ transformants following expression of wild-type I-SceI (dark blue bars labeled “DSB”), K223I (orange bars labeled “SSB”), or D145A (yellow bars labeled “Non-break”) using either of the single or the pair of oligonucleotides to repair the break. All data are presented as the median with range (n≥5). For the specific numerical values see Table S3B-D. (B) Gene correction frequencies by oligonucleotides when an SSB is generated on the “Crick” (left) or “Watson” (right) chromosomal strand. (C) Frequency of transformants in RAD51 (left) or rad51 mutant (right) strains when the SSB is generated on the “Crick” strand. (D) Frequency of transformants following expression of wild-type I-SceI in RAD51 (left) or rad51 mutant (right) strains with final galactose concentrations of 2% (dark blue bars labeled “DSB (2%)”) or 0.02% (light blue bars labeled “DSB (0.02%)”). Wild-type I-SceI strains used: SAS-227 and SAS-228 (“Crick” and RAD51), SAS-281 and SAS-282 (“Watson”), and SAS-235 and SAS-236 (rad51Δ). K223I I-SceI strains used: SAS-229 and SAS-230 (“Crick” and RAD51), SAS-283 and SAS-284 (“Watson”), and SAS-237 and SAS-238 (rad51Δ). D145A I-SceI strains used: SAS-231 and SAS-232 (“Crick” and RAD51), SAS-285 and SAS-286 (“Watson”), and SAS-239 and SAS-240 (rad51Δ).

Mentions: Next, we tested gene correction using DNA oligonucleotides following expression of the different I-SceI variants, in an assay strain in which the genomic TRP5 locus has been disrupted by insertion of the I-SceI recognition site in opposite orientations (Figure 3A). In strains derived from SAS-193, K223I I-SceI can only generate a nick on the “Crick” strand (SAS-229 and SAS-230), while in strains derived from SAS-278, K223 I-SceI can only generate a nick on the “Watson” strand (SAS-283 and SAS-284) (Table S1). DNA oligonucleotides 80-bp in length were designed with homology to either side of the trp5 disruption site such that they could restore the sequence of the TRP5 gene if used as a repair template. Previously, it was shown that oligonucleotides can efficiently transfer genetic modifications to genomic DNA in yeast following generation of a DSB at the targeting locus [10]. Similarly, after expression of wild-type I-SceI in strains SAS-227 and SAS-228 a DSB was generated which was repaired using oligonucleotides (TRP5.80F (F), corresponding to the sense strand of the gene, and TRP5.80R (R), representing the antisense strand) designed to restore the sequence of the disrupted trp5 locus (Table S2). While Trp+ colonies were detected following generation of the DSB even without oligonucleotides, the sequence of the TRP5 locus in these colonies differs from those appearing following recombination with the oligonucleotides and these numbers were subtracted from the counts with oligonucleotides prior to statistical analysis. Specifically, 2 random Trp+ clones deriving from no-oligonucleotide transformation in cells expressing wild-type I-SceI were tested for the presence of the BamHI site. Following PCR amplification of the TRP5 locus using primers TRP5.80F and TRP5.80R, and restriction digestion by BamHI, 2/2 had the PCR product uncut. Differently, 10/10 clones derived from F plus R oligonucleotide transformation in cells expressing wild-type I-SceI or K223I I-SceI had the PCR product cut by BamHI showing that the trp5 allele was repaired by the oligonucleotide sequence (Figure S1). Gene correction following DSB induction was efficient for the complementary pair compared to the non-break control (160-fold increase: p = 0.0022) as well as for the single-stranded F or R oligonucleotide compared to the non-break control (190-fold increase for F: p = 0.0022, or 80-fold increase for R: p = 0.0022, respectively; Figure 3B, left). Similarly, when expression of K223I I-SceI in strains SAS-229 and SAS-230 was used to introduce a break, an increase in recombination frequency was observed following transformation with oligonucleotides F, R, and the F+R pair (up to 9-fold increase for F: p = 0.0022; 8.2-fold for R: p = 0.0022; and 4.5-fold for pair: p = 0.0087, respectively). Recombination using the 80-mers after K223I I-SceI expression was 3-10% as efficient relative to that observed after expression of wild-type I-SceI. Similar results were obtained using the strains containing the “Watson” construct (Figure 3B, right). While fewer colonies arose following generation of the DSB in the “no oligo” control in this construct, likely due to the different distribution of stop codons between the constructs upon end joining, those Trp+ colonies which were detected were subtracted from values with oligonucleotides prior to statistical analysis. Additionally, there was no significant strand bias for gene correction by oligonucleotides following expression of wild-type I-SceI or K223I I-SceI in strains containing either the “Crick” (p≥0.1797) (Figure 3B, left) or the “Watson” (p≥0.3776) (Figure 3B, right) constructs as determined by comparing the frequencies of recombination using the F versus R oligonucleotide.


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 K223I I-SceI break stimulates gene correction by oligonucleotides in yeast.(A) Scheme showing disrupted yeast trp5 chromosomal locus containing the I-SceI recognition sequence (black box). The position of the SSB is indicated (“Nick”) for the “Crick” and “Watson” constructs. Dashed gray lines indicate the complementarity between the F oligonucleotide and the antisense strand of the targeted gene, and between the R oligonucleotide and the sense strand of the targeted gene. (B–D) Frequencies of Trp+ transformants following expression of wild-type I-SceI (dark blue bars labeled “DSB”), K223I (orange bars labeled “SSB”), or D145A (yellow bars labeled “Non-break”) using either of the single or the pair of oligonucleotides to repair the break. All data are presented as the median with range (n≥5). For the specific numerical values see Table S3B-D. (B) Gene correction frequencies by oligonucleotides when an SSB is generated on the “Crick” (left) or “Watson” (right) chromosomal strand. (C) Frequency of transformants in RAD51 (left) or rad51  mutant (right) strains when the SSB is generated on the “Crick” strand. (D) Frequency of transformants following expression of wild-type I-SceI in RAD51 (left) or rad51  mutant (right) strains with final galactose concentrations of 2% (dark blue bars labeled “DSB (2%)”) or 0.02% (light blue bars labeled “DSB (0.02%)”). Wild-type I-SceI strains used: SAS-227 and SAS-228 (“Crick” and RAD51), SAS-281 and SAS-282 (“Watson”), and SAS-235 and SAS-236 (rad51Δ). K223I I-SceI strains used: SAS-229 and SAS-230 (“Crick” and RAD51), SAS-283 and SAS-284 (“Watson”), and SAS-237 and SAS-238 (rad51Δ). D145A I-SceI strains used: SAS-231 and SAS-232 (“Crick” and RAD51), SAS-285 and SAS-286 (“Watson”), and SAS-239 and SAS-240 (rad51Δ).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0088840-g003: A K223I I-SceI break stimulates gene correction by oligonucleotides in yeast.(A) Scheme showing disrupted yeast trp5 chromosomal locus containing the I-SceI recognition sequence (black box). The position of the SSB is indicated (“Nick”) for the “Crick” and “Watson” constructs. Dashed gray lines indicate the complementarity between the F oligonucleotide and the antisense strand of the targeted gene, and between the R oligonucleotide and the sense strand of the targeted gene. (B–D) Frequencies of Trp+ transformants following expression of wild-type I-SceI (dark blue bars labeled “DSB”), K223I (orange bars labeled “SSB”), or D145A (yellow bars labeled “Non-break”) using either of the single or the pair of oligonucleotides to repair the break. All data are presented as the median with range (n≥5). For the specific numerical values see Table S3B-D. (B) Gene correction frequencies by oligonucleotides when an SSB is generated on the “Crick” (left) or “Watson” (right) chromosomal strand. (C) Frequency of transformants in RAD51 (left) or rad51 mutant (right) strains when the SSB is generated on the “Crick” strand. (D) Frequency of transformants following expression of wild-type I-SceI in RAD51 (left) or rad51 mutant (right) strains with final galactose concentrations of 2% (dark blue bars labeled “DSB (2%)”) or 0.02% (light blue bars labeled “DSB (0.02%)”). Wild-type I-SceI strains used: SAS-227 and SAS-228 (“Crick” and RAD51), SAS-281 and SAS-282 (“Watson”), and SAS-235 and SAS-236 (rad51Δ). K223I I-SceI strains used: SAS-229 and SAS-230 (“Crick” and RAD51), SAS-283 and SAS-284 (“Watson”), and SAS-237 and SAS-238 (rad51Δ). D145A I-SceI strains used: SAS-231 and SAS-232 (“Crick” and RAD51), SAS-285 and SAS-286 (“Watson”), and SAS-239 and SAS-240 (rad51Δ).
Mentions: Next, we tested gene correction using DNA oligonucleotides following expression of the different I-SceI variants, in an assay strain in which the genomic TRP5 locus has been disrupted by insertion of the I-SceI recognition site in opposite orientations (Figure 3A). In strains derived from SAS-193, K223I I-SceI can only generate a nick on the “Crick” strand (SAS-229 and SAS-230), while in strains derived from SAS-278, K223 I-SceI can only generate a nick on the “Watson” strand (SAS-283 and SAS-284) (Table S1). DNA oligonucleotides 80-bp in length were designed with homology to either side of the trp5 disruption site such that they could restore the sequence of the TRP5 gene if used as a repair template. Previously, it was shown that oligonucleotides can efficiently transfer genetic modifications to genomic DNA in yeast following generation of a DSB at the targeting locus [10]. Similarly, after expression of wild-type I-SceI in strains SAS-227 and SAS-228 a DSB was generated which was repaired using oligonucleotides (TRP5.80F (F), corresponding to the sense strand of the gene, and TRP5.80R (R), representing the antisense strand) designed to restore the sequence of the disrupted trp5 locus (Table S2). While Trp+ colonies were detected following generation of the DSB even without oligonucleotides, the sequence of the TRP5 locus in these colonies differs from those appearing following recombination with the oligonucleotides and these numbers were subtracted from the counts with oligonucleotides prior to statistical analysis. Specifically, 2 random Trp+ clones deriving from no-oligonucleotide transformation in cells expressing wild-type I-SceI were tested for the presence of the BamHI site. Following PCR amplification of the TRP5 locus using primers TRP5.80F and TRP5.80R, and restriction digestion by BamHI, 2/2 had the PCR product uncut. Differently, 10/10 clones derived from F plus R oligonucleotide transformation in cells expressing wild-type I-SceI or K223I I-SceI had the PCR product cut by BamHI showing that the trp5 allele was repaired by the oligonucleotide sequence (Figure S1). Gene correction following DSB induction was efficient for the complementary pair compared to the non-break control (160-fold increase: p = 0.0022) as well as for the single-stranded F or R oligonucleotide compared to the non-break control (190-fold increase for F: p = 0.0022, or 80-fold increase for R: p = 0.0022, respectively; Figure 3B, left). Similarly, when expression of K223I I-SceI in strains SAS-229 and SAS-230 was used to introduce a break, an increase in recombination frequency was observed following transformation with oligonucleotides F, R, and the F+R pair (up to 9-fold increase for F: p = 0.0022; 8.2-fold for R: p = 0.0022; and 4.5-fold for pair: p = 0.0087, respectively). Recombination using the 80-mers after K223I I-SceI expression was 3-10% as efficient relative to that observed after expression of wild-type I-SceI. Similar results were obtained using the strains containing the “Watson” construct (Figure 3B, right). While fewer colonies arose following generation of the DSB in the “no oligo” control in this construct, likely due to the different distribution of stop codons between the constructs upon end joining, those Trp+ colonies which were detected were subtracted from values with oligonucleotides prior to statistical analysis. Additionally, there was no significant strand bias for gene correction by oligonucleotides following expression of wild-type I-SceI or K223I I-SceI in strains containing either the “Crick” (p≥0.1797) (Figure 3B, left) or the “Watson” (p≥0.3776) (Figure 3B, right) constructs as determined by comparing the frequencies of recombination using the F versus R oligonucleotide.

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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