Limits...
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.


TALE sequence and structure.(a) Comparison between the amino acid sequences of the first 4 TAL-repeats of TALE with/without the periodically mutated repeats. The sequences of TAL-repeats without (CT-TALE, upper) and with the periodical mutations (VT-TALE, lower) are presented. RVDs are shaded in cyan. Non-RVD residues at the 4th and 32nd positions are presented as bold letters with different colours for the mutation sites. The secondary structures are presented at the bottom. (b) Ribbon representations of a single-TAL repeat (left) and the DNA bound TALE protein (right) (PDBID: 3V6T14). The first short helix (helix a) and the second long helix (helix b) are coloured red and green, respectively. RVD and the periodical mutation sites are noted in cyan and magenta, respectively, with the side chains shown as a stick representation. In the right panel, RVDs and the mutation sites are also displayed. DNA is also presented in orange. Figures were prepared using PyMOL (DeLano Scientific, San Carlos, CA). (c) Schematic representation of CT-TALE (upper) and VT-TALE (lower). TAL-repeats are represented as yellow boxes. Non-canonical pseudo-repeats12 and the last half repeats30 are presented as dashed boxes and white boxes, respectively. Amino acid types of non-RVD residues at the 4th and 32nd positions in each repeat are given as letters; mutated sites are coloured.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5121632&req=5

f1: TALE sequence and structure.(a) Comparison between the amino acid sequences of the first 4 TAL-repeats of TALE with/without the periodically mutated repeats. The sequences of TAL-repeats without (CT-TALE, upper) and with the periodical mutations (VT-TALE, lower) are presented. RVDs are shaded in cyan. Non-RVD residues at the 4th and 32nd positions are presented as bold letters with different colours for the mutation sites. The secondary structures are presented at the bottom. (b) Ribbon representations of a single-TAL repeat (left) and the DNA bound TALE protein (right) (PDBID: 3V6T14). The first short helix (helix a) and the second long helix (helix b) are coloured red and green, respectively. RVD and the periodical mutation sites are noted in cyan and magenta, respectively, with the side chains shown as a stick representation. In the right panel, RVDs and the mutation sites are also displayed. DNA is also presented in orange. Figures were prepared using PyMOL (DeLano Scientific, San Carlos, CA). (c) Schematic representation of CT-TALE (upper) and VT-TALE (lower). TAL-repeats are represented as yellow boxes. Non-canonical pseudo-repeats12 and the last half repeats30 are presented as dashed boxes and white boxes, respectively. Amino acid types of non-RVD residues at the 4th and 32nd positions in each repeat are given as letters; mutated sites are coloured.

Mentions: Transcription activator-like effector (TALE) proteins secreted by plant bacterial pathogens bind to specific host promoters12345. The DNA binding region of the TALE protein constitutes a unique array of repeats (TAL-repeat). These 34-residue repeats share a highly conserved sequence, except for two central residues at positions 12 and 13, which are denoted as the repeat variable diresidue, RVD2678 (Fig. 1a and b). In binding to DNA, an array of TALE RVDs ensure sequence specificity2678. The code relating RVDs to target DNA bases is established67. This modularity is exploited to generate TALE proteins tailored to recognise prescribed DNA sequences of interest67. An artificial fusion protein composed of a custom TAL-repeat array and the sequence independent FokI nuclease domain that functions as a dimer9 is called TALEN, and this protein is used widely as a genome editing tool1011.


Non-RVD mutations that enhance the dynamics of the TAL repeat array along the superhelical axis improve TALEN genome editing efficacy
TALE sequence and structure.(a) Comparison between the amino acid sequences of the first 4 TAL-repeats of TALE with/without the periodically mutated repeats. The sequences of TAL-repeats without (CT-TALE, upper) and with the periodical mutations (VT-TALE, lower) are presented. RVDs are shaded in cyan. Non-RVD residues at the 4th and 32nd positions are presented as bold letters with different colours for the mutation sites. The secondary structures are presented at the bottom. (b) Ribbon representations of a single-TAL repeat (left) and the DNA bound TALE protein (right) (PDBID: 3V6T14). The first short helix (helix a) and the second long helix (helix b) are coloured red and green, respectively. RVD and the periodical mutation sites are noted in cyan and magenta, respectively, with the side chains shown as a stick representation. In the right panel, RVDs and the mutation sites are also displayed. DNA is also presented in orange. Figures were prepared using PyMOL (DeLano Scientific, San Carlos, CA). (c) Schematic representation of CT-TALE (upper) and VT-TALE (lower). TAL-repeats are represented as yellow boxes. Non-canonical pseudo-repeats12 and the last half repeats30 are presented as dashed boxes and white boxes, respectively. Amino acid types of non-RVD residues at the 4th and 32nd positions in each repeat are given as letters; mutated sites are coloured.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: TALE sequence and structure.(a) Comparison between the amino acid sequences of the first 4 TAL-repeats of TALE with/without the periodically mutated repeats. The sequences of TAL-repeats without (CT-TALE, upper) and with the periodical mutations (VT-TALE, lower) are presented. RVDs are shaded in cyan. Non-RVD residues at the 4th and 32nd positions are presented as bold letters with different colours for the mutation sites. The secondary structures are presented at the bottom. (b) Ribbon representations of a single-TAL repeat (left) and the DNA bound TALE protein (right) (PDBID: 3V6T14). The first short helix (helix a) and the second long helix (helix b) are coloured red and green, respectively. RVD and the periodical mutation sites are noted in cyan and magenta, respectively, with the side chains shown as a stick representation. In the right panel, RVDs and the mutation sites are also displayed. DNA is also presented in orange. Figures were prepared using PyMOL (DeLano Scientific, San Carlos, CA). (c) Schematic representation of CT-TALE (upper) and VT-TALE (lower). TAL-repeats are represented as yellow boxes. Non-canonical pseudo-repeats12 and the last half repeats30 are presented as dashed boxes and white boxes, respectively. Amino acid types of non-RVD residues at the 4th and 32nd positions in each repeat are given as letters; mutated sites are coloured.
Mentions: Transcription activator-like effector (TALE) proteins secreted by plant bacterial pathogens bind to specific host promoters12345. The DNA binding region of the TALE protein constitutes a unique array of repeats (TAL-repeat). These 34-residue repeats share a highly conserved sequence, except for two central residues at positions 12 and 13, which are denoted as the repeat variable diresidue, RVD2678 (Fig. 1a and b). In binding to DNA, an array of TALE RVDs ensure sequence specificity2678. The code relating RVDs to target DNA bases is established67. This modularity is exploited to generate TALE proteins tailored to recognise prescribed DNA sequences of interest67. An artificial fusion protein composed of a custom TAL-repeat array and the sequence independent FokI nuclease domain that functions as a dimer9 is called TALEN, and this protein is used widely as a genome editing tool1011.

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.