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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

The efficacy of MNs on their chromosomal target is poorly correlated with the activity of the nucleases in extrachromosomal assays. Throughout the figure, 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. (a) Comparison between the extrachromosomal SSA assay in CHO-KI and the TM assay in HEK293 cells. Thirty-seven MNs cleaving 39 targets were tested in both assays (Table 1). Correlation was also made with a smaller sample of 18 MNs (black circles), which are the same as the 18 MNs characterized in the HGT assay (Figure 3b), and displayed on Figure 3c. Vertical grey lines represent the activity levels of I-SceIm (left line) and Rag1m (right line). (b) Correlation between the extrachromosomal SSA assays in 293-H cells and the TM assay in 293-H cells. (c) Correlation between the extrachromosomal SSA assays in CHO cells and the HGT assay in 293-H cells. (d) Correlation between the extrachromosomal SSA assays in 293-H cells and the HGT assay in 293-H cells.
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gks268-F4: The efficacy of MNs on their chromosomal target is poorly correlated with the activity of the nucleases in extrachromosomal assays. Throughout the figure, 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. (a) Comparison between the extrachromosomal SSA assay in CHO-KI and the TM assay in HEK293 cells. Thirty-seven MNs cleaving 39 targets were tested in both assays (Table 1). Correlation was also made with a smaller sample of 18 MNs (black circles), which are the same as the 18 MNs characterized in the HGT assay (Figure 3b), and displayed on Figure 3c. Vertical grey lines represent the activity levels of I-SceIm (left line) and Rag1m (right line). (b) Correlation between the extrachromosomal SSA assays in 293-H cells and the TM assay in 293-H cells. (c) Correlation between the extrachromosomal SSA assays in CHO cells and the HGT assay in 293-H cells. (d) Correlation between the extrachromosomal SSA assays in 293-H cells and the HGT assay in 293-H cells.

Mentions: aFor statistics of Figures 3 and 4, results for CAPNS1 target are from CAPNS1m.


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)

The efficacy of MNs on their chromosomal target is poorly correlated with the activity of the nucleases in extrachromosomal assays. Throughout the figure, 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. (a) Comparison between the extrachromosomal SSA assay in CHO-KI and the TM assay in HEK293 cells. Thirty-seven MNs cleaving 39 targets were tested in both assays (Table 1). Correlation was also made with a smaller sample of 18 MNs (black circles), which are the same as the 18 MNs characterized in the HGT assay (Figure 3b), and displayed on Figure 3c. Vertical grey lines represent the activity levels of I-SceIm (left line) and Rag1m (right line). (b) Correlation between the extrachromosomal SSA assays in 293-H cells and the TM assay in 293-H cells. (c) Correlation between the extrachromosomal SSA assays in CHO cells and the HGT assay in 293-H cells. (d) Correlation between the extrachromosomal SSA assays in 293-H cells and the HGT assay in 293-H cells.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks268-F4: The efficacy of MNs on their chromosomal target is poorly correlated with the activity of the nucleases in extrachromosomal assays. Throughout the figure, 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. (a) Comparison between the extrachromosomal SSA assay in CHO-KI and the TM assay in HEK293 cells. Thirty-seven MNs cleaving 39 targets were tested in both assays (Table 1). Correlation was also made with a smaller sample of 18 MNs (black circles), which are the same as the 18 MNs characterized in the HGT assay (Figure 3b), and displayed on Figure 3c. Vertical grey lines represent the activity levels of I-SceIm (left line) and Rag1m (right line). (b) Correlation between the extrachromosomal SSA assays in 293-H cells and the TM assay in 293-H cells. (c) Correlation between the extrachromosomal SSA assays in CHO cells and the HGT assay in 293-H cells. (d) Correlation between the extrachromosomal SSA assays in 293-H cells and the HGT assay in 293-H cells.
Mentions: aFor statistics of Figures 3 and 4, results for CAPNS1 target are from CAPNS1m.

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