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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 that selectively broaden mCreI cleavage specificity. (A and D) Native mCreI cleaves −6C and to a lesser extent −6T at low protein concentrations, whereas Design 22 permits nearly equal cleavage of the −6G design target base pair as well as −6C or T. (B and C) The 26Q sidechain in native mCreI contacts the guanine complementary to −6C (B), but cannot make productive contacts with −6G either from 77I or 26Q (C). (E) In Design 22, 77R contacts both the −6G design target base as well as the adjacent −7A. (F) In the presence of the native −6C:G base pair, 77R pivots to make less favorable contacts to −7A and −8A that permit cleavage but with little base selectivity. Designs with comparably broadened specificities are referred to as Class III designs.
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Figure 6: Designs that selectively broaden mCreI cleavage specificity. (A and D) Native mCreI cleaves −6C and to a lesser extent −6T at low protein concentrations, whereas Design 22 permits nearly equal cleavage of the −6G design target base pair as well as −6C or T. (B and C) The 26Q sidechain in native mCreI contacts the guanine complementary to −6C (B), but cannot make productive contacts with −6G either from 77I or 26Q (C). (E) In Design 22, 77R contacts both the −6G design target base as well as the adjacent −7A. (F) In the presence of the native −6C:G base pair, 77R pivots to make less favorable contacts to −7A and −8A that permit cleavage but with little base selectivity. Designs with comparably broadened specificities are referred to as Class III designs.

Mentions: Class III designs included four mCreI variants with broader cleavage specificities relative to native mCreI, and an average RD-predicted specificity of 65%. All four Class III designs cleaved both their design base pair and the native base pair at the design position. Design 22, for −6G, is representative of Class III designs (Figure 6). Designs with lower overall specificity may be practically useful when a desired novel specificity can be achieved despite retaining the ability to cleave the native target site base pair.Figure 6.


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

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

Designs that selectively broaden mCreI cleavage specificity. (A and D) Native mCreI cleaves −6C and to a lesser extent −6T at low protein concentrations, whereas Design 22 permits nearly equal cleavage of the −6G design target base pair as well as −6C or T. (B and C) The 26Q sidechain in native mCreI contacts the guanine complementary to −6C (B), but cannot make productive contacts with −6G either from 77I or 26Q (C). (E) In Design 22, 77R contacts both the −6G design target base as well as the adjacent −7A. (F) In the presence of the native −6C:G base pair, 77R pivots to make less favorable contacts to −7A and −8A that permit cleavage but with little base selectivity. Designs with comparably broadened specificities are referred to as Class III designs.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 6: Designs that selectively broaden mCreI cleavage specificity. (A and D) Native mCreI cleaves −6C and to a lesser extent −6T at low protein concentrations, whereas Design 22 permits nearly equal cleavage of the −6G design target base pair as well as −6C or T. (B and C) The 26Q sidechain in native mCreI contacts the guanine complementary to −6C (B), but cannot make productive contacts with −6G either from 77I or 26Q (C). (E) In Design 22, 77R contacts both the −6G design target base as well as the adjacent −7A. (F) In the presence of the native −6C:G base pair, 77R pivots to make less favorable contacts to −7A and −8A that permit cleavage but with little base selectivity. Designs with comparably broadened specificities are referred to as Class III designs.
Mentions: Class III designs included four mCreI variants with broader cleavage specificities relative to native mCreI, and an average RD-predicted specificity of 65%. All four Class III designs cleaved both their design base pair and the native base pair at the design position. Design 22, for −6G, is representative of Class III designs (Figure 6). Designs with lower overall specificity may be practically useful when a desired novel specificity can be achieved despite retaining the ability to cleave the native target site base pair.Figure 6.

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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