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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 altered cleavage specificity at a target site position. (A) Native mCreI preferentially cleaves target sites with −5G, and to a lesser extent −5A followed by −5C or T. (B) Recognition of the −5 position by native mCreI is mediated by 2 contacts made by residue 68R to −5G, and non-polar contact of 24I with the complementary C. (C) Design 28 cleaves −5C to near-completion even at 20 nM, with minor activity on −5T. (D) Recognition of −5C in Design 28 is mediated by 24I→K that contacts to the complementary G at −5, and by 68R→T that prevents potentially deleterious contacts with −5C. Designs with comparably altered specificities are referred to as Class II designs.
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Figure 5: Designs with altered cleavage specificity at a target site position. (A) Native mCreI preferentially cleaves target sites with −5G, and to a lesser extent −5A followed by −5C or T. (B) Recognition of the −5 position by native mCreI is mediated by 2 contacts made by residue 68R to −5G, and non-polar contact of 24I with the complementary C. (C) Design 28 cleaves −5C to near-completion even at 20 nM, with minor activity on −5T. (D) Recognition of −5C in Design 28 is mediated by 24I→K that contacts to the complementary G at −5, and by 68R→T that prevents potentially deleterious contacts with −5C. Designs with comparably altered specificities are referred to as Class II designs.

Mentions: Class II designs included seven mCreI variants that cleaved design base pairs with specificities comparable to mCreI. The average RD-predicted specificity of Class II enzymes was 77%, and all seven Class II designs were approximately as active as native mCreI (Table 1; Supplementary Figure S1). Design 28, a representative example of Class II designs (Figure 5), preferentially cleaved −5C and to a lesser extent −5T. Native mCreI, in contrast, preferentially cleaved −5G and to a lesser extent −5A (Supplementary Figure S1 top row).Figure 5.


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

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

Designs with altered cleavage specificity at a target site position. (A) Native mCreI preferentially cleaves target sites with −5G, and to a lesser extent −5A followed by −5C or T. (B) Recognition of the −5 position by native mCreI is mediated by 2 contacts made by residue 68R to −5G, and non-polar contact of 24I with the complementary C. (C) Design 28 cleaves −5C to near-completion even at 20 nM, with minor activity on −5T. (D) Recognition of −5C in Design 28 is mediated by 24I→K that contacts to the complementary G at −5, and by 68R→T that prevents potentially deleterious contacts with −5C. Designs with comparably altered specificities are referred to as Class II designs.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 5: Designs with altered cleavage specificity at a target site position. (A) Native mCreI preferentially cleaves target sites with −5G, and to a lesser extent −5A followed by −5C or T. (B) Recognition of the −5 position by native mCreI is mediated by 2 contacts made by residue 68R to −5G, and non-polar contact of 24I with the complementary C. (C) Design 28 cleaves −5C to near-completion even at 20 nM, with minor activity on −5T. (D) Recognition of −5C in Design 28 is mediated by 24I→K that contacts to the complementary G at −5, and by 68R→T that prevents potentially deleterious contacts with −5C. Designs with comparably altered specificities are referred to as Class II designs.
Mentions: Class II designs included seven mCreI variants that cleaved design base pairs with specificities comparable to mCreI. The average RD-predicted specificity of Class II enzymes was 77%, and all seven Class II designs were approximately as active as native mCreI (Table 1; Supplementary Figure S1). Design 28, a representative example of Class II designs (Figure 5), preferentially cleaved −5C and to a lesser extent −5T. Native mCreI, in contrast, preferentially cleaved −5G and to a lesser extent −5A (Supplementary Figure S1 top row).Figure 5.

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