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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.


Enhanced superhelical motion by the non-RVD mutations to TALEN.(a) The superhelical amplitude motion of VT-TALE is greater in magnitude compared with that of CT-TALE. The model TALE structures in different conformations occurred in the molecular dynamics simulation are located on the left to the schematic drawings. The canonical TAL-repeats, N-terminal atypical repeats and C-terminal half repeats are indicated as yellow, grey and white boxes, respectively. (b) The number of TAL-repeats in VT-TALE (lower) that engage in DNA binding is greater than that observed for CT-TALE (upper). The DNA-bound TAL-repeats are represented by orange boxes, and the free TAL-repeats are drawn as yellow boxes. The DNA bases specifically recognised by the TAL-repeats are presented as red bars. The modelled TALE structures in the complex with DNA are located on the left of the schematic drawings for the complexes having a different number of TAL-repeats engaged in the DNA binding. (c) The spatial restriction of the FokI domains by the extended TAL-repeat interaction to DNA in VT-TALE (lower) could facilitate FokI dimerisation more readily than that observed for CT-TALE (upper). FokI monomers are depicted as cyan semicircles. The arrows schematically represent the magnitudes of the structural dynamics of the TAL-repeats not bound to DNA and the FokI domain associated with the C-terminal end of the TAL-repeat array.
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f5: Enhanced superhelical motion by the non-RVD mutations to TALEN.(a) The superhelical amplitude motion of VT-TALE is greater in magnitude compared with that of CT-TALE. The model TALE structures in different conformations occurred in the molecular dynamics simulation are located on the left to the schematic drawings. The canonical TAL-repeats, N-terminal atypical repeats and C-terminal half repeats are indicated as yellow, grey and white boxes, respectively. (b) The number of TAL-repeats in VT-TALE (lower) that engage in DNA binding is greater than that observed for CT-TALE (upper). The DNA-bound TAL-repeats are represented by orange boxes, and the free TAL-repeats are drawn as yellow boxes. The DNA bases specifically recognised by the TAL-repeats are presented as red bars. The modelled TALE structures in the complex with DNA are located on the left of the schematic drawings for the complexes having a different number of TAL-repeats engaged in the DNA binding. (c) The spatial restriction of the FokI domains by the extended TAL-repeat interaction to DNA in VT-TALE (lower) could facilitate FokI dimerisation more readily than that observed for CT-TALE (upper). FokI monomers are depicted as cyan semicircles. The arrows schematically represent the magnitudes of the structural dynamics of the TAL-repeats not bound to DNA and the FokI domain associated with the C-terminal end of the TAL-repeat array.

Mentions: TALE has a superhelical motion, which is evident in several lines of experimental1415 and theoretical1617 evidence, which makes TALE adopt various conformations in different sizes along its superhelical axis. VT-TALE exhibited a greater magnitude in the superhelical motion and can subsequently take a wider range of structures over CT-TALE (Fig. 5a).


Non-RVD mutations that enhance the dynamics of the TAL repeat array along the superhelical axis improve TALEN genome editing efficacy
Enhanced superhelical motion by the non-RVD mutations to TALEN.(a) The superhelical amplitude motion of VT-TALE is greater in magnitude compared with that of CT-TALE. The model TALE structures in different conformations occurred in the molecular dynamics simulation are located on the left to the schematic drawings. The canonical TAL-repeats, N-terminal atypical repeats and C-terminal half repeats are indicated as yellow, grey and white boxes, respectively. (b) The number of TAL-repeats in VT-TALE (lower) that engage in DNA binding is greater than that observed for CT-TALE (upper). The DNA-bound TAL-repeats are represented by orange boxes, and the free TAL-repeats are drawn as yellow boxes. The DNA bases specifically recognised by the TAL-repeats are presented as red bars. The modelled TALE structures in the complex with DNA are located on the left of the schematic drawings for the complexes having a different number of TAL-repeats engaged in the DNA binding. (c) The spatial restriction of the FokI domains by the extended TAL-repeat interaction to DNA in VT-TALE (lower) could facilitate FokI dimerisation more readily than that observed for CT-TALE (upper). FokI monomers are depicted as cyan semicircles. The arrows schematically represent the magnitudes of the structural dynamics of the TAL-repeats not bound to DNA and the FokI domain associated with the C-terminal end of the TAL-repeat array.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Enhanced superhelical motion by the non-RVD mutations to TALEN.(a) The superhelical amplitude motion of VT-TALE is greater in magnitude compared with that of CT-TALE. The model TALE structures in different conformations occurred in the molecular dynamics simulation are located on the left to the schematic drawings. The canonical TAL-repeats, N-terminal atypical repeats and C-terminal half repeats are indicated as yellow, grey and white boxes, respectively. (b) The number of TAL-repeats in VT-TALE (lower) that engage in DNA binding is greater than that observed for CT-TALE (upper). The DNA-bound TAL-repeats are represented by orange boxes, and the free TAL-repeats are drawn as yellow boxes. The DNA bases specifically recognised by the TAL-repeats are presented as red bars. The modelled TALE structures in the complex with DNA are located on the left of the schematic drawings for the complexes having a different number of TAL-repeats engaged in the DNA binding. (c) The spatial restriction of the FokI domains by the extended TAL-repeat interaction to DNA in VT-TALE (lower) could facilitate FokI dimerisation more readily than that observed for CT-TALE (upper). FokI monomers are depicted as cyan semicircles. The arrows schematically represent the magnitudes of the structural dynamics of the TAL-repeats not bound to DNA and the FokI domain associated with the C-terminal end of the TAL-repeat array.
Mentions: TALE has a superhelical motion, which is evident in several lines of experimental1415 and theoretical1617 evidence, which makes TALE adopt various conformations in different sizes along its superhelical axis. VT-TALE exhibited a greater magnitude in the superhelical motion and can subsequently take a wider range of structures over CT-TALE (Fig. 5a).

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.