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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.


Inter-repeat hydrogen bonds among the TAL-repeats.(a) Snapshots of the CT- and VT-TALE models in the extended forms sampled at 50 ns in the MD trajectories. The structures are viewed from the C-terminal side of the superhelical structure and its 90-degree rotated position. For the four TAL-repeats in the centre of the array, the side chains of the residues positioned at 4 and 5 in each repeat of CT- and VT-TALEs are drawn as ball and stick representations. Residue at position 4, a non-RVD residue, in each TAL-repeat is marked in purple. For details of the modelling and simulation, see the Supplementary Information. (b) Distances between HE21/HE22 in Q-5 and OD1/OD2 in D-4 or OE1/OE2 in E-4 in the structures in the MD trajectory (corresponding to Trial 1 in Supplementary Fig. S4); every atomic pair is considered. (c) Schematic representation of the inter-repeat hydrogen bonds. Helices a and b and RVD are presented as magenta boxes, green boxes and cyan lines, respectively. The 4th and 5th amino acids in each TAL-repeat are represented by short lines. Hydrogen bonds are indicated by orange lines. In VT-TALE, two of the inter-repeat hydrogen bonds are not formed in a unit with four TAL-repeats.
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f3: Inter-repeat hydrogen bonds among the TAL-repeats.(a) Snapshots of the CT- and VT-TALE models in the extended forms sampled at 50 ns in the MD trajectories. The structures are viewed from the C-terminal side of the superhelical structure and its 90-degree rotated position. For the four TAL-repeats in the centre of the array, the side chains of the residues positioned at 4 and 5 in each repeat of CT- and VT-TALEs are drawn as ball and stick representations. Residue at position 4, a non-RVD residue, in each TAL-repeat is marked in purple. For details of the modelling and simulation, see the Supplementary Information. (b) Distances between HE21/HE22 in Q-5 and OD1/OD2 in D-4 or OE1/OE2 in E-4 in the structures in the MD trajectory (corresponding to Trial 1 in Supplementary Fig. S4); every atomic pair is considered. (c) Schematic representation of the inter-repeat hydrogen bonds. Helices a and b and RVD are presented as magenta boxes, green boxes and cyan lines, respectively. The 4th and 5th amino acids in each TAL-repeat are represented by short lines. Hydrogen bonds are indicated by orange lines. In VT-TALE, two of the inter-repeat hydrogen bonds are not formed in a unit with four TAL-repeats.

Mentions: Residues D-4 and Q-5 in the adjacent repeats were close to each other in the TAL-repeat array (Fig. 3a); the hyphenated residue name refers to the position within each TAL-repeat (Fig. 1a). As seen in the MD trajectories for CT-TALE, the hydrogen bonding donor and acceptor side chain atoms of D-4 and Q-5 stochastically become close to enable the inter-repeat hydrogen bond in the nanosecond time regime (Fig. 3b). The probability for the inter-repeat hydrogen bonding among the 11 sites in the extended CT-TALE (MD trajectory during 40–50 ns) was 12.1% (Supplementary Table S2).


Non-RVD mutations that enhance the dynamics of the TAL repeat array along the superhelical axis improve TALEN genome editing efficacy
Inter-repeat hydrogen bonds among the TAL-repeats.(a) Snapshots of the CT- and VT-TALE models in the extended forms sampled at 50 ns in the MD trajectories. The structures are viewed from the C-terminal side of the superhelical structure and its 90-degree rotated position. For the four TAL-repeats in the centre of the array, the side chains of the residues positioned at 4 and 5 in each repeat of CT- and VT-TALEs are drawn as ball and stick representations. Residue at position 4, a non-RVD residue, in each TAL-repeat is marked in purple. For details of the modelling and simulation, see the Supplementary Information. (b) Distances between HE21/HE22 in Q-5 and OD1/OD2 in D-4 or OE1/OE2 in E-4 in the structures in the MD trajectory (corresponding to Trial 1 in Supplementary Fig. S4); every atomic pair is considered. (c) Schematic representation of the inter-repeat hydrogen bonds. Helices a and b and RVD are presented as magenta boxes, green boxes and cyan lines, respectively. The 4th and 5th amino acids in each TAL-repeat are represented by short lines. Hydrogen bonds are indicated by orange lines. In VT-TALE, two of the inter-repeat hydrogen bonds are not formed in a unit with four TAL-repeats.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Inter-repeat hydrogen bonds among the TAL-repeats.(a) Snapshots of the CT- and VT-TALE models in the extended forms sampled at 50 ns in the MD trajectories. The structures are viewed from the C-terminal side of the superhelical structure and its 90-degree rotated position. For the four TAL-repeats in the centre of the array, the side chains of the residues positioned at 4 and 5 in each repeat of CT- and VT-TALEs are drawn as ball and stick representations. Residue at position 4, a non-RVD residue, in each TAL-repeat is marked in purple. For details of the modelling and simulation, see the Supplementary Information. (b) Distances between HE21/HE22 in Q-5 and OD1/OD2 in D-4 or OE1/OE2 in E-4 in the structures in the MD trajectory (corresponding to Trial 1 in Supplementary Fig. S4); every atomic pair is considered. (c) Schematic representation of the inter-repeat hydrogen bonds. Helices a and b and RVD are presented as magenta boxes, green boxes and cyan lines, respectively. The 4th and 5th amino acids in each TAL-repeat are represented by short lines. Hydrogen bonds are indicated by orange lines. In VT-TALE, two of the inter-repeat hydrogen bonds are not formed in a unit with four TAL-repeats.
Mentions: Residues D-4 and Q-5 in the adjacent repeats were close to each other in the TAL-repeat array (Fig. 3a); the hyphenated residue name refers to the position within each TAL-repeat (Fig. 1a). As seen in the MD trajectories for CT-TALE, the hydrogen bonding donor and acceptor side chain atoms of D-4 and Q-5 stochastically become close to enable the inter-repeat hydrogen bond in the nanosecond time regime (Fig. 3b). The probability for the inter-repeat hydrogen bonding among the 11 sites in the extended CT-TALE (MD trajectory during 40–50 ns) was 12.1% (Supplementary Table S2).

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.