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Comprehensive computational design of mCreI homing endonuclease cleavage specificity for genome engineering.

Ulge UY, Baker DA, Monnat RJ - Nucleic Acids Res. (2011)

Bottom Line: Homing endonucleases (HEs) cleave long (∼ 20 bp) DNA target sites with high site specificity to catalyze the lateral transfer of parasitic DNA elements.Experimental verification of a range of these designs demonstrated that over 2/3 (24 of 35 designs, 69%) had the intended new site specificity, and that 14 of the 15 attempted specificity shifts (93%) were achieved.These results demonstrate the feasibility of using structure-based computational design to engineer HE variants with novel target site specificities to facilitate genome engineering.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Howard Hughes Medical InstituteUniversity of Washington, Box 357705, Seattle, WA 98195, USA.

ABSTRACT
Homing endonucleases (HEs) cleave long (∼ 20 bp) DNA target sites with high site specificity to catalyze the lateral transfer of parasitic DNA elements. In order to determine whether comprehensive computational design could be used as a general strategy to engineer new HE target site specificities, we used RosettaDesign (RD) to generate 3200 different variants of the mCreI LAGLIDADG HE towards 16 different base pair positions in the 22 bp mCreI target site. Experimental verification of a range of these designs demonstrated that over 2/3 (24 of 35 designs, 69%) had the intended new site specificity, and that 14 of the 15 attempted specificity shifts (93%) were achieved. These results demonstrate the feasibility of using structure-based computational design to engineer HE variants with novel target site specificities to facilitate genome engineering.

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Designs with enhanced cleavage specificity at a degenerate target site position. (A) Native mCreI cleaves all four base pair possibilities at the −8 target site position. (B) This lack of specificity reflects the presence of a single water-mediated bond from 28K to −8A. (C) Design 11, in contrast, cleaves only −8G even at high enzyme concentrations. This and comparable designs with enhanced specificities are referred to as Class I designs (see text). (D) The enhanced specificity of Design 11 appears to reflect the ability of residue substitutions to specify a G:C base pair at this position: 40T→R donates two hydrogen bonds to guanine, and 28K→D accepts a hydrogen bond from the complementary −8C. Neither of these interactions was possible with the native target site A:T base pair. Native amino acid residues and the native target site base pair are shown in yellow, design residue substitutions and variant target site base pairs in green, and water molecules as blue spheres (not to scale). The structure of the native enzyme bound to native target site DNA is from the co-crystal structure of I-CreI determined by Chevalier and colleagues (PDB ID 1G9Y). The corresponding structures for designs were computationally generated molecular models.
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Figure 4: Designs with enhanced cleavage specificity at a degenerate target site position. (A) Native mCreI cleaves all four base pair possibilities at the −8 target site position. (B) This lack of specificity reflects the presence of a single water-mediated bond from 28K to −8A. (C) Design 11, in contrast, cleaves only −8G even at high enzyme concentrations. This and comparable designs with enhanced specificities are referred to as Class I designs (see text). (D) The enhanced specificity of Design 11 appears to reflect the ability of residue substitutions to specify a G:C base pair at this position: 40T→R donates two hydrogen bonds to guanine, and 28K→D accepts a hydrogen bond from the complementary −8C. Neither of these interactions was possible with the native target site A:T base pair. Native amino acid residues and the native target site base pair are shown in yellow, design residue substitutions and variant target site base pairs in green, and water molecules as blue spheres (not to scale). The structure of the native enzyme bound to native target site DNA is from the co-crystal structure of I-CreI determined by Chevalier and colleagues (PDB ID 1G9Y). The corresponding structures for designs were computationally generated molecular models.

Mentions: The mCreI design successes summarized above represent three classes of outcome with different combinations of specificity and activity that each may be useful for specific engineering applications. Class I, containing four designs, had the highest average RD-predicted specificity of 87% (Figure 7; data not shown). These designs were more specific than mCreI, especially at high-protein concentrations, but were generally less active than native mCreI (Table 1, Supplementary Figure S1). Of note, our prior analyses of mCreI and mMsoI (30) emphasized that even modest levels of catalytic activity are sufficient to promote in vivo cleavage-dependent recombination in human cells. Molecular modeling of Class I designs indicated two different strategies that conferred high specificity: suppressing cleavage of a native base pair while favoring cleavage of an alternative base pair and designing toward the native base pair while suppressing cleavage of other tolerated base pairs. Design 11 is an example of the first of these strategies (Figure 4).Figure 4.


Comprehensive computational design of mCreI homing endonuclease cleavage specificity for genome engineering.

Ulge UY, Baker DA, Monnat RJ - Nucleic Acids Res. (2011)

Designs with enhanced cleavage specificity at a degenerate target site position. (A) Native mCreI cleaves all four base pair possibilities at the −8 target site position. (B) This lack of specificity reflects the presence of a single water-mediated bond from 28K to −8A. (C) Design 11, in contrast, cleaves only −8G even at high enzyme concentrations. This and comparable designs with enhanced specificities are referred to as Class I designs (see text). (D) The enhanced specificity of Design 11 appears to reflect the ability of residue substitutions to specify a G:C base pair at this position: 40T→R donates two hydrogen bonds to guanine, and 28K→D accepts a hydrogen bond from the complementary −8C. Neither of these interactions was possible with the native target site A:T base pair. Native amino acid residues and the native target site base pair are shown in yellow, design residue substitutions and variant target site base pairs in green, and water molecules as blue spheres (not to scale). The structure of the native enzyme bound to native target site DNA is from the co-crystal structure of I-CreI determined by Chevalier and colleagues (PDB ID 1G9Y). The corresponding structures for designs were computationally generated molecular models.
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Related In: Results  -  Collection

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Figure 4: Designs with enhanced cleavage specificity at a degenerate target site position. (A) Native mCreI cleaves all four base pair possibilities at the −8 target site position. (B) This lack of specificity reflects the presence of a single water-mediated bond from 28K to −8A. (C) Design 11, in contrast, cleaves only −8G even at high enzyme concentrations. This and comparable designs with enhanced specificities are referred to as Class I designs (see text). (D) The enhanced specificity of Design 11 appears to reflect the ability of residue substitutions to specify a G:C base pair at this position: 40T→R donates two hydrogen bonds to guanine, and 28K→D accepts a hydrogen bond from the complementary −8C. Neither of these interactions was possible with the native target site A:T base pair. Native amino acid residues and the native target site base pair are shown in yellow, design residue substitutions and variant target site base pairs in green, and water molecules as blue spheres (not to scale). The structure of the native enzyme bound to native target site DNA is from the co-crystal structure of I-CreI determined by Chevalier and colleagues (PDB ID 1G9Y). The corresponding structures for designs were computationally generated molecular models.
Mentions: The mCreI design successes summarized above represent three classes of outcome with different combinations of specificity and activity that each may be useful for specific engineering applications. Class I, containing four designs, had the highest average RD-predicted specificity of 87% (Figure 7; data not shown). These designs were more specific than mCreI, especially at high-protein concentrations, but were generally less active than native mCreI (Table 1, Supplementary Figure S1). Of note, our prior analyses of mCreI and mMsoI (30) emphasized that even modest levels of catalytic activity are sufficient to promote in vivo cleavage-dependent recombination in human cells. Molecular modeling of Class I designs indicated two different strategies that conferred high specificity: suppressing cleavage of a native base pair while favoring cleavage of an alternative base pair and designing toward the native base pair while suppressing cleavage of other tolerated base pairs. Design 11 is an example of the first of these strategies (Figure 4).Figure 4.

Bottom Line: Homing endonucleases (HEs) cleave long (∼ 20 bp) DNA target sites with high site specificity to catalyze the lateral transfer of parasitic DNA elements.Experimental verification of a range of these designs demonstrated that over 2/3 (24 of 35 designs, 69%) had the intended new site specificity, and that 14 of the 15 attempted specificity shifts (93%) were achieved.These results demonstrate the feasibility of using structure-based computational design to engineer HE variants with novel target site specificities to facilitate genome engineering.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Howard Hughes Medical InstituteUniversity of Washington, Box 357705, Seattle, WA 98195, USA.

ABSTRACT
Homing endonucleases (HEs) cleave long (∼ 20 bp) DNA target sites with high site specificity to catalyze the lateral transfer of parasitic DNA elements. In order to determine whether comprehensive computational design could be used as a general strategy to engineer new HE target site specificities, we used RosettaDesign (RD) to generate 3200 different variants of the mCreI LAGLIDADG HE towards 16 different base pair positions in the 22 bp mCreI target site. Experimental verification of a range of these designs demonstrated that over 2/3 (24 of 35 designs, 69%) had the intended new site specificity, and that 14 of the 15 attempted specificity shifts (93%) were achieved. These results demonstrate the feasibility of using structure-based computational design to engineer HE variants with novel target site specificities to facilitate genome engineering.

Show MeSH
Related in: MedlinePlus