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Chromosomal context and epigenetic mechanisms control the efficacy of genome editing by rare-cutting designer endonucleases.

Daboussi F, Zaslavskiy M, Poirot L, Loperfido M, Gouble A, Guyot V, Leduc S, Galetto R, Grizot S, Oficjalska D, Perez C, Delacôte F, Dupuy A, Chion-Sotinel I, Le Clerre D, Lebuhotel C, Danos O, Lemaire F, Oussedik K, Cédrone F, Epinat JC, Smith J, Yáñez-Muñoz RJ, Dickson G, Popplewell L, Koo T, VandenDriessche T, Chuah MK, Duclert A, Duchateau P, Pâques F - Nucleic Acids Res. (2012)

Bottom Line: In contrast, the efficiency of targeted mutagenesis or homologous gene targeting at a given chromosomal locus does not correlate with the activity of individual endonucleases on transiently transfected substrates.Finally, we demonstrate that chromatin accessibility modulates the efficacy of rare-cutting endonucleases, accounting for strong position effects.Thus, chromosomal context and epigenetic mechanisms may play a major role in the efficiency rare-cutting endonuclease-induced genome engineering.

View Article: PubMed Central - PubMed

Affiliation: CELLECTIS S.A., Paris, France.

ABSTRACT
The ability to specifically engineer the genome of living cells at precise locations using rare-cutting designer endonucleases has broad implications for biotechnology and medicine, particularly for functional genomics, transgenics and gene therapy. However, the potential impact of chromosomal context and epigenetics on designer endonuclease-mediated genome editing is poorly understood. To address this question, we conducted a comprehensive analysis on the efficacy of 37 endonucleases derived from the quintessential I-CreI meganuclease that were specifically designed to cleave 39 different genomic targets. The analysis revealed that the efficiency of targeted mutagenesis at a given chromosomal locus is predictive of that of homologous gene targeting. Consequently, a strong genome-wide correlation was apparent between the efficiency of targeted mutagenesis (≤ 0.1% to ≈ 6%) with that of homologous gene targeting (≤ 0.1% to ≈ 15%). In contrast, the efficiency of targeted mutagenesis or homologous gene targeting at a given chromosomal locus does not correlate with the activity of individual endonucleases on transiently transfected substrates. Finally, we demonstrate that chromatin accessibility modulates the efficacy of rare-cutting endonucleases, accounting for strong position effects. Thus, chromosomal context and epigenetic mechanisms may play a major role in the efficiency rare-cutting endonuclease-induced genome engineering.

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Related in: MedlinePlus

Characterization of MNs properties using cell-based assays. (a) An extrachromosomal assay for the characterization of MN activity in mammalian cells. Briefly, MN expressing vectors and reporter vector are co-transfected into CHO-KI or 293-H cells. Upon cleavage of the target site, tandem repeat recombination by SSA between two truncated copies of the LacZ gene restores a functional β-galactosidase gene, which can be monitored by standard assays. For each MN, a dose response is performed and AUC is used as a quantitative measure of MN activity. (b) Example of read out of the extrachromosomal assay described in panel. The activity of the CLS4076m, I-SceIm and Rag1m MNs is featured as an example. AUC is used as a quantitative measure of MN activity in this type of extrachromosomal assay. For CLS4076, AUC is the area in grey. (c) Monitoring of MN expression by western blotting. 293-H cells and CHO-K1 were transfected with a plasmid-encoding MN. The MN expression level was monitored by western blot. (d) Quantification of MN expression level. The MN expression level from western blot (c) was quantified and normalized to β-tubulin signal. These ratios were plotted for both cell lines and statistical analysis was performed. r, Pearson coefficient (linear correlation coefficient); ρ, Spearman coefficient (non-linear correlation coefficient); N, sample size; P, probability of finding a given correlation when the underlying variables are not correlated. (e) Comparison between the extra-chromosomal SSA assays in CHO-KI and HEK293 cells. Sixteen MNs were tested in both assays (Table 1). Dotted grey curves represent 95% confidence interval for the regression line. r, Pearson coefficient (linear correlation coefficient); ρ, Spearman coefficient (non-linear correlation coefficient); N, sample size; P, probability of finding a given correlation when the underlying variables are not correlated.
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gks268-F2: Characterization of MNs properties using cell-based assays. (a) An extrachromosomal assay for the characterization of MN activity in mammalian cells. Briefly, MN expressing vectors and reporter vector are co-transfected into CHO-KI or 293-H cells. Upon cleavage of the target site, tandem repeat recombination by SSA between two truncated copies of the LacZ gene restores a functional β-galactosidase gene, which can be monitored by standard assays. For each MN, a dose response is performed and AUC is used as a quantitative measure of MN activity. (b) Example of read out of the extrachromosomal assay described in panel. The activity of the CLS4076m, I-SceIm and Rag1m MNs is featured as an example. AUC is used as a quantitative measure of MN activity in this type of extrachromosomal assay. For CLS4076, AUC is the area in grey. (c) Monitoring of MN expression by western blotting. 293-H cells and CHO-K1 were transfected with a plasmid-encoding MN. The MN expression level was monitored by western blot. (d) Quantification of MN expression level. The MN expression level from western blot (c) was quantified and normalized to β-tubulin signal. These ratios were plotted for both cell lines and statistical analysis was performed. r, Pearson coefficient (linear correlation coefficient); ρ, Spearman coefficient (non-linear correlation coefficient); N, sample size; P, probability of finding a given correlation when the underlying variables are not correlated. (e) Comparison between the extra-chromosomal SSA assays in CHO-KI and HEK293 cells. Sixteen MNs were tested in both assays (Table 1). Dotted grey curves represent 95% confidence interval for the regression line. r, Pearson coefficient (linear correlation coefficient); ρ, Spearman coefficient (non-linear correlation coefficient); N, sample size; P, probability of finding a given correlation when the underlying variables are not correlated.

Mentions: Area under the curve (AUC) score is used to quantify (i) the activity of MN in extrachromosomal assays in CHO-KI and 293-H cells; (ii) cell survival in the toxicity assay; (iii) chromatin resistance to microccocal nuclease digestion at a given locus. Examples are given in Figure 2b and Supplementary Figure S2c. The classical approach based on the use of log-normal model (30) showed a very good fitting quality for both survival curves and activity curves (after normalization with respect to the maximal activity level). All the statistics done in this study are summarized in Supplementary Table S6.


Chromosomal context and epigenetic mechanisms control the efficacy of genome editing by rare-cutting designer endonucleases.

Daboussi F, Zaslavskiy M, Poirot L, Loperfido M, Gouble A, Guyot V, Leduc S, Galetto R, Grizot S, Oficjalska D, Perez C, Delacôte F, Dupuy A, Chion-Sotinel I, Le Clerre D, Lebuhotel C, Danos O, Lemaire F, Oussedik K, Cédrone F, Epinat JC, Smith J, Yáñez-Muñoz RJ, Dickson G, Popplewell L, Koo T, VandenDriessche T, Chuah MK, Duclert A, Duchateau P, Pâques F - Nucleic Acids Res. (2012)

Characterization of MNs properties using cell-based assays. (a) An extrachromosomal assay for the characterization of MN activity in mammalian cells. Briefly, MN expressing vectors and reporter vector are co-transfected into CHO-KI or 293-H cells. Upon cleavage of the target site, tandem repeat recombination by SSA between two truncated copies of the LacZ gene restores a functional β-galactosidase gene, which can be monitored by standard assays. For each MN, a dose response is performed and AUC is used as a quantitative measure of MN activity. (b) Example of read out of the extrachromosomal assay described in panel. The activity of the CLS4076m, I-SceIm and Rag1m MNs is featured as an example. AUC is used as a quantitative measure of MN activity in this type of extrachromosomal assay. For CLS4076, AUC is the area in grey. (c) Monitoring of MN expression by western blotting. 293-H cells and CHO-K1 were transfected with a plasmid-encoding MN. The MN expression level was monitored by western blot. (d) Quantification of MN expression level. The MN expression level from western blot (c) was quantified and normalized to β-tubulin signal. These ratios were plotted for both cell lines and statistical analysis was performed. r, Pearson coefficient (linear correlation coefficient); ρ, Spearman coefficient (non-linear correlation coefficient); N, sample size; P, probability of finding a given correlation when the underlying variables are not correlated. (e) Comparison between the extra-chromosomal SSA assays in CHO-KI and HEK293 cells. Sixteen MNs were tested in both assays (Table 1). Dotted grey curves represent 95% confidence interval for the regression line. r, Pearson coefficient (linear correlation coefficient); ρ, Spearman coefficient (non-linear correlation coefficient); N, sample size; P, probability of finding a given correlation when the underlying variables are not correlated.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3401453&req=5

gks268-F2: Characterization of MNs properties using cell-based assays. (a) An extrachromosomal assay for the characterization of MN activity in mammalian cells. Briefly, MN expressing vectors and reporter vector are co-transfected into CHO-KI or 293-H cells. Upon cleavage of the target site, tandem repeat recombination by SSA between two truncated copies of the LacZ gene restores a functional β-galactosidase gene, which can be monitored by standard assays. For each MN, a dose response is performed and AUC is used as a quantitative measure of MN activity. (b) Example of read out of the extrachromosomal assay described in panel. The activity of the CLS4076m, I-SceIm and Rag1m MNs is featured as an example. AUC is used as a quantitative measure of MN activity in this type of extrachromosomal assay. For CLS4076, AUC is the area in grey. (c) Monitoring of MN expression by western blotting. 293-H cells and CHO-K1 were transfected with a plasmid-encoding MN. The MN expression level was monitored by western blot. (d) Quantification of MN expression level. The MN expression level from western blot (c) was quantified and normalized to β-tubulin signal. These ratios were plotted for both cell lines and statistical analysis was performed. r, Pearson coefficient (linear correlation coefficient); ρ, Spearman coefficient (non-linear correlation coefficient); N, sample size; P, probability of finding a given correlation when the underlying variables are not correlated. (e) Comparison between the extra-chromosomal SSA assays in CHO-KI and HEK293 cells. Sixteen MNs were tested in both assays (Table 1). Dotted grey curves represent 95% confidence interval for the regression line. r, Pearson coefficient (linear correlation coefficient); ρ, Spearman coefficient (non-linear correlation coefficient); N, sample size; P, probability of finding a given correlation when the underlying variables are not correlated.
Mentions: Area under the curve (AUC) score is used to quantify (i) the activity of MN in extrachromosomal assays in CHO-KI and 293-H cells; (ii) cell survival in the toxicity assay; (iii) chromatin resistance to microccocal nuclease digestion at a given locus. Examples are given in Figure 2b and Supplementary Figure S2c. The classical approach based on the use of log-normal model (30) showed a very good fitting quality for both survival curves and activity curves (after normalization with respect to the maximal activity level). All the statistics done in this study are summarized in Supplementary Table S6.

Bottom Line: In contrast, the efficiency of targeted mutagenesis or homologous gene targeting at a given chromosomal locus does not correlate with the activity of individual endonucleases on transiently transfected substrates.Finally, we demonstrate that chromatin accessibility modulates the efficacy of rare-cutting endonucleases, accounting for strong position effects.Thus, chromosomal context and epigenetic mechanisms may play a major role in the efficiency rare-cutting endonuclease-induced genome engineering.

View Article: PubMed Central - PubMed

Affiliation: CELLECTIS S.A., Paris, France.

ABSTRACT
The ability to specifically engineer the genome of living cells at precise locations using rare-cutting designer endonucleases has broad implications for biotechnology and medicine, particularly for functional genomics, transgenics and gene therapy. However, the potential impact of chromosomal context and epigenetics on designer endonuclease-mediated genome editing is poorly understood. To address this question, we conducted a comprehensive analysis on the efficacy of 37 endonucleases derived from the quintessential I-CreI meganuclease that were specifically designed to cleave 39 different genomic targets. The analysis revealed that the efficiency of targeted mutagenesis at a given chromosomal locus is predictive of that of homologous gene targeting. Consequently, a strong genome-wide correlation was apparent between the efficiency of targeted mutagenesis (≤ 0.1% to ≈ 6%) with that of homologous gene targeting (≤ 0.1% to ≈ 15%). In contrast, the efficiency of targeted mutagenesis or homologous gene targeting at a given chromosomal locus does not correlate with the activity of individual endonucleases on transiently transfected substrates. Finally, we demonstrate that chromatin accessibility modulates the efficacy of rare-cutting endonucleases, accounting for strong position effects. Thus, chromosomal context and epigenetic mechanisms may play a major role in the efficiency rare-cutting endonuclease-induced genome engineering.

Show MeSH
Related in: MedlinePlus