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Comprehensive analysis of the specificity of transcription activator-like effector nucleases

Juillerat, Alexandre, Dubois, Gwendoline, Valton, Julien, Thomas, Séverine, Stella, Stefano, Maréchal, Alan, Langevin, Stéphanie, Benomari, Nassima, Bertonati, Claudia, Silva, George H., Daboussi, Fayza, Epinat, Jean-Charles, Montoya, Guillermo, Duclert, Aymeric, Duchateau, Philippe·2014·Nucleic Acids Research
DOI 10.1093/nar/gku155PMC PMC4005648
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Figure 1. Gene disruption-relative activities of collection of TALEN at the integrated GFP locus. (A) Schematic representation of the WT GFP TALEN on the chromosomal target. (B) Collections of TALE used at the chromosomal GFP locus derived by mutation of the right DNA binding domain. X represents any of the four, namely, NI, HD, NN and NG RVD. Positions are numbered relative to the first thymine of the target (T0). (C) Influence of the number of mismatches on the GFP disruption. Activity ratio between the mismatched and the WT TALEN is represented on a boxplot, indicating the median (thick bar), quartiles (box) and extreme values (r = −0.68, P = 4e−16). Mismatches are defined relative to the NI:A, HD:C, NN:G and NG:T codes. The size of the sample is indicated in brackets. (D) Boxplot representation, including the median (thick bar), quartiles (box) and extreme values, of the activity ratio between the mismatched and the WT TALEN in function of the collections for one mismatch. P = 0.08 (Kruskal–Wallis test). (E) Same as for (D) but for two mismatches. P = 4e−4. (F) Same as in (D) but for three mismatches. P = 2e−4.
Figure 1. Gene disruption-relative activities of collection of TALEN at the integrated GFP locus. (A) Schematic representation of the WT GFP TALEN on the chromosomal target. (B) Collections of TALE used at the chromosomal GFP locus derived by mutation of the right DNA binding domain. X represents any of the four, namely, NI, HD, NN and NG RVD. Positions are numbered relative to the first thymine of the target (T0). (C) Influence of the number of mismatches on the GFP disruption. Activity ratio between the mismatched and the WT TALEN is represented on a boxplot, indicating the median (thick bar), quartiles (box) and extreme values (r = −0.68, P = 4e−16). Mismatches are defined relative to the NI:A, HD:C, NN:G and NG:T codes. The size of the sample is indicated in brackets. (D) Boxplot representation, including the median (thick bar), quartiles (box) and extreme values, of the activity ratio between the mismatched and the WT TALEN in function of the collections for one mismatch. P = 0.08 (Kruskal–Wallis test). (E) Same as for (D) but for two mismatches. P = 4e−4. (F) Same as in (D) but for three mismatches. P = 2e−4.CC BY-NC · PubMed Central
Figure 2. Design of experimental setup and optimal TALEN array length for the nuclease activity screening. (A) Activities of 52 TALEN pairs measured in vivo, in function of the repeat array length. Error bars represent standard errors. (B) Activities of 15 TALEN pairs on targets randomized in their positions N and N-1. Error bars represent standard errors. An analysis of variance demonstrates a significant effect of the TALEN length on specificity, position N: r = 0.68, P = 4e−6 (Kruskal–Wallis test); position N-1: r = 0.7, P = 7e−6). The yeast activity assay is based on the single-strand annealing (SSA) pathway used after the creation of a DSB by the TALEN in the target sequence. Target sites were designed to allow TALEN use in the homodimer format. All TALEN pairs showed a significant activity. The number of TALEN/target pairs for each class is indicated in brackets.
Figure 2. Design of experimental setup and optimal TALEN array length for the nuclease activity screening. (A) Activities of 52 TALEN pairs measured in vivo, in function of the repeat array length. Error bars represent standard errors. (B) Activities of 15 TALEN pairs on targets randomized in their positions N and N-1. Error bars represent standard errors. An analysis of variance demonstrates a significant effect of the TALEN length on specificity, position N: r = 0.68, P = 4e−6 (Kruskal–Wallis test); position N-1: r = 0.7, P = 7e−6). The yeast activity assay is based on the single-strand annealing (SSA) pathway used after the creation of a DSB by the TALEN in the target sequence. Target sites were designed to allow TALEN use in the homodimer format. All TALEN pairs showed a significant activity. The number of TALEN/target pairs for each class is indicated in brackets.CC BY-NC · PubMed Central
Figure 3. Setup of collections used in the large-scale experiments and graphical representation of activity results from Collection 6. (A) Collections of TALE and targets used for the study in yeast, where X represents any of the four NI, HD, NN and NG RVD and N any of the four A, C, G and T bases. Collections 6–8 are composed of arrays containing 9.5 repeats and Collection 9 is composed of arrays of 18.5. The TALEN collections were used in the homodimer format. (B) Heatmap showing the activity of the 64 TALEN of Collection 6 on the 64 corresponding targets. The outer line of text of the target (abscissa) represents the first nucleotide of the NNN triplet, the middle line of text represents the second nucleotide and the innermost line of text represents the third nucleotide. Likewise for the RVD array (XXX) on the ordinate. Red corresponds to maximum activity, whereas blue corresponds to no activity. The diagonal with framed squares represents the NI:A, HD:C, NN:G and NG:T pairings. In the case of a perfect one to one RVD/nucleotide association code, activity should be recovered on the diagonal only.
Figure 3. Setup of collections used in the large-scale experiments and graphical representation of activity results from Collection 6. (A) Collections of TALE and targets used for the study in yeast, where X represents any of the four NI, HD, NN and NG RVD and N any of the four A, C, G and T bases. Collections 6–8 are composed of arrays containing 9.5 repeats and Collection 9 is composed of arrays of 18.5. The TALEN collections were used in the homodimer format. (B) Heatmap showing the activity of the 64 TALEN of Collection 6 on the 64 corresponding targets. The outer line of text of the target (abscissa) represents the first nucleotide of the NNN triplet, the middle line of text represents the second nucleotide and the innermost line of text represents the third nucleotide. Likewise for the RVD array (XXX) on the ordinate. Red corresponds to maximum activity, whereas blue corresponds to no activity. The diagonal with framed squares represents the NI:A, HD:C, NN:G and NG:T pairings. In the case of a perfect one to one RVD/nucleotide association code, activity should be recovered on the diagonal only.CC BY-NC · PubMed Central
Figure 4. Graphical representation of RVD specificities. (A) Specificity measured for positions 1–7. The level of gray (black = 1, white = 0) represents the relative activity compared with the HD:C, NG:T NI:A and NN:G RVD/nucleotide pairing code. (B) Average specificity of the four HD, NG, NI and NN RVDs on the first 7 positions. (C) Logo representation of the global specificity matrix. Logo was generated using WebLogo (http://weblogo.berkeley.edu/logo.cgi). Values for relative specificities are presented in Supplementary Table S4.
Figure 4. Graphical representation of RVD specificities. (A) Specificity measured for positions 1–7. The level of gray (black = 1, white = 0) represents the relative activity compared with the HD:C, NG:T NI:A and NN:G RVD/nucleotide pairing code. (B) Average specificity of the four HD, NG, NI and NN RVDs on the first 7 positions. (C) Logo representation of the global specificity matrix. Logo was generated using WebLogo (http://weblogo.berkeley.edu/logo.cgi). Values for relative specificities are presented in Supplementary Table S4.CC BY-NC · PubMed Central
Figure 5. Effect of target mutations on TALEN activity in mammalian cells. (A) Correlation between experimental relative activities represented by the percentage of GFP-negative cells in the mammalian gene-targeting assay and scoring using the matrix presented in Figure 4B (Collections 1, 2 and 3 are represented in red: r = 0.81, P = 4e−16, and Collections 4 and 5 are represented in blue: r = 0.84, P = 3e−12). Linear regressions are presented for both subsets and 95% confidence intervals are represented by dashed lines. (B) Pie chart representation of the percentage of TALEN composed of 15.5 RVDs that will have, in the human genome, potential off-site targets containing no, one, two, three or four and more mismatches when considering a test set of 15 000 putative TALEN. All possible combinations of half TALEN (left + right, left + left, right + right) with a spacer length ranging from 9 to 30 bp were taken into account. (C) A collection of 33 targets comprising two AvrBs3 target sequences facing each other on both DNA strand with spacer length (between the two targets) ranging from 5 to 40 bp were designed and assayed in the CHO-K1 SSA assay to determine the optimal cleavage conditions. Targets containing a spacer of 21 and 35 bp were absent from the study.
Figure 5. Effect of target mutations on TALEN activity in mammalian cells. (A) Correlation between experimental relative activities represented by the percentage of GFP-negative cells in the mammalian gene-targeting assay and scoring using the matrix presented in Figure 4B (Collections 1, 2 and 3 are represented in red: r = 0.81, P = 4e−16, and Collections 4 and 5 are represented in blue: r = 0.84, P = 3e−12). Linear regressions are presented for both subsets and 95% confidence intervals are represented by dashed lines. (B) Pie chart representation of the percentage of TALEN composed of 15.5 RVDs that will have, in the human genome, potential off-site targets containing no, one, two, three or four and more mismatches when considering a test set of 15 000 putative TALEN. All possible combinations of half TALEN (left + right, left + left, right + right) with a spacer length ranging from 9 to 30 bp were taken into account. (C) A collection of 33 targets comprising two AvrBs3 target sequences facing each other on both DNA strand with spacer length (between the two targets) ranging from 5 to 40 bp were designed and assayed in the CHO-K1 SSA assay to determine the optimal cleavage conditions. Targets containing a spacer of 21 and 35 bp were absent from the study.CC BY-NC · PubMed Central

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Metadata

Authors
Juillerat, A., Dubois, G., Valton, J., Thomas, S., Stella, S., Maréchal, A., Langevin, S., Benomari, N., Bertonati, C., Silva, G. H., Daboussi, F., Epinat, J.-C., Montoya, G., Duclert, A., & Duchateau, P.
Venue
Nucleic Acids Research
Publisher
Oxford University Press (OUP)
Published
2014-02-24
Volume
42
Issue
8
Pages
5390-5402
ISSN
  • 1362-4962
  • 0305-1048
DOI
10.1093/nar/gku155
PMC
PMC4005648
Cited by
93
References
2
Language
en
Licence
creativecommons.org/licenses/by-nc/3.0/ (vor, 953 d)

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