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Non-RVD mutations that enhance the dynamics of the TAL repeat array along the superhelical axis improve TALEN genome editing efficacy

View Article: PubMed Central - PubMed

ABSTRACT

Transcription activator-like effector (TALE) nuclease (TALEN) is widely used as a tool in genome editing. The DNA binding part of TALEN consists of a tandem array of TAL-repeats that form a right-handed superhelix. Each TAL-repeat recognises a specific base by the repeat variable diresidue (RVD) at positions 12 and 13. TALEN comprising the TAL-repeats with periodic mutations to residues at positions 4 and 32 (non-RVD sites) in each repeat (VT-TALE) exhibits increased efficacy in genome editing compared with a counterpart without the mutations (CT-TALE). The molecular basis for the elevated efficacy is unknown. In this report, comparison of the physicochemical properties between CT- and VT-TALEs revealed that VT-TALE has a larger amplitude motion along the superhelical axis (superhelical motion) compared with CT-TALE. The greater superhelical motion in VT-TALE enabled more TAL-repeats to engage in the target sequence recognition compared with CT-TALE. The extended sequence recognition by the TAL-repeats improves site specificity with limiting the spatial distribution of FokI domains to facilitate their dimerization at the desired site. Molecular dynamics simulations revealed that the non-RVD mutations alter inter-repeat hydrogen bonding to amplify the superhelical motion of VT-TALE. The TALEN activity is associated with the inter-repeat hydrogen bonding among the TAL repeats.

No MeSH data available.


Comparison of physicochemical properties between CT-TALE and VT-TALE.(a) Analyses of size exclusion chromatograms of TALEs. Calibration of the column was performed using a standard globular protein set (GE Healthcare), and the chromatogram is presented in green. Stokes radii of the standard proteins are labelled (Å unit)27. The chromatograms of CT-TALE and VT-TALE are indicated in blue and red, respectively. Estimated Stokes radii of CT-TALE and VT-TALE are shown in parentheses. (b) CD spectra of TALEs. CD spectra of CT-TALE and VT-TALE are presented in blue and red, respectively. (c) Size distributions of CT- and VT-TALE. Particle size distributions of CT- and VT-TALE are presented in blue and red, respectively. Estimated Z-average sizes were 48.3 ± 0.7 Å and 44.7 ± 1.6 Å, respectively. (d) DSC thermograms of the CT- and VT-TALEs. Experimental data are shown in black, and fits for individual and composite fits are indicated in red. (upper) CT-TALE: T1m = 61.71 ± 0.02 °C; T2m = 63.41 ± 0.01 °C, (lower) VT-TALE: Tm = 52.07 ± 0.01 °C.
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f2: Comparison of physicochemical properties between CT-TALE and VT-TALE.(a) Analyses of size exclusion chromatograms of TALEs. Calibration of the column was performed using a standard globular protein set (GE Healthcare), and the chromatogram is presented in green. Stokes radii of the standard proteins are labelled (Å unit)27. The chromatograms of CT-TALE and VT-TALE are indicated in blue and red, respectively. Estimated Stokes radii of CT-TALE and VT-TALE are shown in parentheses. (b) CD spectra of TALEs. CD spectra of CT-TALE and VT-TALE are presented in blue and red, respectively. (c) Size distributions of CT- and VT-TALE. Particle size distributions of CT- and VT-TALE are presented in blue and red, respectively. Estimated Z-average sizes were 48.3 ± 0.7 Å and 44.7 ± 1.6 Å, respectively. (d) DSC thermograms of the CT- and VT-TALEs. Experimental data are shown in black, and fits for individual and composite fits are indicated in red. (upper) CT-TALE: T1m = 61.71 ± 0.02 °C; T2m = 63.41 ± 0.01 °C, (lower) VT-TALE: Tm = 52.07 ± 0.01 °C.

Mentions: The CT-TALE and VT-TALE proteins used in this study have 16.5 TAL repeats and four atypical repeats at the N-terminus (Fig. 1c). Both TALEs share the same array of RVDs (Fig. 1c). SEC analysis showed that CT-TALE is larger in size than VT-TALE; the Stokes radii (RS) for CT-TALE and VT-TALE were estimated to be 46 Å and 42 Å, respectively (Fig. 2a). The radius of a globular protein with the same molecular weight, 80 kDa, is 28 Å21. The elucidated radii for CT- and VT-TALEs demonstrate that they adopt elongated shapes, as established by the crystal structures of TALEs15212223.


Non-RVD mutations that enhance the dynamics of the TAL repeat array along the superhelical axis improve TALEN genome editing efficacy
Comparison of physicochemical properties between CT-TALE and VT-TALE.(a) Analyses of size exclusion chromatograms of TALEs. Calibration of the column was performed using a standard globular protein set (GE Healthcare), and the chromatogram is presented in green. Stokes radii of the standard proteins are labelled (Å unit)27. The chromatograms of CT-TALE and VT-TALE are indicated in blue and red, respectively. Estimated Stokes radii of CT-TALE and VT-TALE are shown in parentheses. (b) CD spectra of TALEs. CD spectra of CT-TALE and VT-TALE are presented in blue and red, respectively. (c) Size distributions of CT- and VT-TALE. Particle size distributions of CT- and VT-TALE are presented in blue and red, respectively. Estimated Z-average sizes were 48.3 ± 0.7 Å and 44.7 ± 1.6 Å, respectively. (d) DSC thermograms of the CT- and VT-TALEs. Experimental data are shown in black, and fits for individual and composite fits are indicated in red. (upper) CT-TALE: T1m = 61.71 ± 0.02 °C; T2m = 63.41 ± 0.01 °C, (lower) VT-TALE: Tm = 52.07 ± 0.01 °C.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5121632&req=5

f2: Comparison of physicochemical properties between CT-TALE and VT-TALE.(a) Analyses of size exclusion chromatograms of TALEs. Calibration of the column was performed using a standard globular protein set (GE Healthcare), and the chromatogram is presented in green. Stokes radii of the standard proteins are labelled (Å unit)27. The chromatograms of CT-TALE and VT-TALE are indicated in blue and red, respectively. Estimated Stokes radii of CT-TALE and VT-TALE are shown in parentheses. (b) CD spectra of TALEs. CD spectra of CT-TALE and VT-TALE are presented in blue and red, respectively. (c) Size distributions of CT- and VT-TALE. Particle size distributions of CT- and VT-TALE are presented in blue and red, respectively. Estimated Z-average sizes were 48.3 ± 0.7 Å and 44.7 ± 1.6 Å, respectively. (d) DSC thermograms of the CT- and VT-TALEs. Experimental data are shown in black, and fits for individual and composite fits are indicated in red. (upper) CT-TALE: T1m = 61.71 ± 0.02 °C; T2m = 63.41 ± 0.01 °C, (lower) VT-TALE: Tm = 52.07 ± 0.01 °C.
Mentions: The CT-TALE and VT-TALE proteins used in this study have 16.5 TAL repeats and four atypical repeats at the N-terminus (Fig. 1c). Both TALEs share the same array of RVDs (Fig. 1c). SEC analysis showed that CT-TALE is larger in size than VT-TALE; the Stokes radii (RS) for CT-TALE and VT-TALE were estimated to be 46 Å and 42 Å, respectively (Fig. 2a). The radius of a globular protein with the same molecular weight, 80 kDa, is 28 Å21. The elucidated radii for CT- and VT-TALEs demonstrate that they adopt elongated shapes, as established by the crystal structures of TALEs15212223.

View Article: PubMed Central - PubMed

ABSTRACT

Transcription activator-like effector (TALE) nuclease (TALEN) is widely used as a tool in genome editing. The DNA binding part of TALEN consists of a tandem array of TAL-repeats that form a right-handed superhelix. Each TAL-repeat recognises a specific base by the repeat variable diresidue (RVD) at positions 12 and 13. TALEN comprising the TAL-repeats with periodic mutations to residues at positions 4 and 32 (non-RVD sites) in each repeat (VT-TALE) exhibits increased efficacy in genome editing compared with a counterpart without the mutations (CT-TALE). The molecular basis for the elevated efficacy is unknown. In this report, comparison of the physicochemical properties between CT- and VT-TALEs revealed that VT-TALE has a larger amplitude motion along the superhelical axis (superhelical motion) compared with CT-TALE. The greater superhelical motion in VT-TALE enabled more TAL-repeats to engage in the target sequence recognition compared with CT-TALE. The extended sequence recognition by the TAL-repeats improves site specificity with limiting the spatial distribution of FokI domains to facilitate their dimerization at the desired site. Molecular dynamics simulations revealed that the non-RVD mutations alter inter-repeat hydrogen bonding to amplify the superhelical motion of VT-TALE. The TALEN activity is associated with the inter-repeat hydrogen bonding among the TAL repeats.

No MeSH data available.