Limits...
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.

Show MeSH
Predicted and experimentally determined cleavage specificities of successful novel mCreI variants. Fourteen designs are shown color coded by Class of outcome and as a function of their RD-predicted and experimentally determined cleavage specificities (see text). Designs predicted to be more specific typically had higher observed specificities, although the relationship between predicted and observed specificities was modest (R2 value of ∼0.5). Observed specificities were calculated by quantifying the cleavage product intensity in gel images (Supplementary Figure S1), then dividing the intensity of the intended cleavage band by the sum of intensities of all cleavage products at the most specific enzyme concentration (usually 20 nM) (see Supplementary Table S1 for quantified cleavage band intensities). Again, as in Figure 3, a design with novel, but unintended, specificity for −8C has been excluded from the plot.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3105429&req=5

Figure 7: Predicted and experimentally determined cleavage specificities of successful novel mCreI variants. Fourteen designs are shown color coded by Class of outcome and as a function of their RD-predicted and experimentally determined cleavage specificities (see text). Designs predicted to be more specific typically had higher observed specificities, although the relationship between predicted and observed specificities was modest (R2 value of ∼0.5). Observed specificities were calculated by quantifying the cleavage product intensity in gel images (Supplementary Figure S1), then dividing the intensity of the intended cleavage band by the sum of intensities of all cleavage products at the most specific enzyme concentration (usually 20 nM) (see Supplementary Table S1 for quantified cleavage band intensities). Again, as in Figure 3, a design with novel, but unintended, specificity for −8C has been excluded from the plot.

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)

Predicted and experimentally determined cleavage specificities of successful novel mCreI variants. Fourteen designs are shown color coded by Class of outcome and as a function of their RD-predicted and experimentally determined cleavage specificities (see text). Designs predicted to be more specific typically had higher observed specificities, although the relationship between predicted and observed specificities was modest (R2 value of ∼0.5). Observed specificities were calculated by quantifying the cleavage product intensity in gel images (Supplementary Figure S1), then dividing the intensity of the intended cleavage band by the sum of intensities of all cleavage products at the most specific enzyme concentration (usually 20 nM) (see Supplementary Table S1 for quantified cleavage band intensities). Again, as in Figure 3, a design with novel, but unintended, specificity for −8C has been excluded from the plot.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105429&req=5

Figure 7: Predicted and experimentally determined cleavage specificities of successful novel mCreI variants. Fourteen designs are shown color coded by Class of outcome and as a function of their RD-predicted and experimentally determined cleavage specificities (see text). Designs predicted to be more specific typically had higher observed specificities, although the relationship between predicted and observed specificities was modest (R2 value of ∼0.5). Observed specificities were calculated by quantifying the cleavage product intensity in gel images (Supplementary Figure S1), then dividing the intensity of the intended cleavage band by the sum of intensities of all cleavage products at the most specific enzyme concentration (usually 20 nM) (see Supplementary Table S1 for quantified cleavage band intensities). Again, as in Figure 3, a design with novel, but unintended, specificity for −8C has been excluded from the plot.
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