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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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In vitro cleavage assay. (A) In vitro ‘bar code’ cleavage assay developed to determine simultaneously cleavage specificity and activity for all four base pair possibilities at single target site positions in a single tube/single gel lane assay. Four primer pairs were used to amplify cleavage substrates from target site plasmids with the target site at the center of the resulting PCR fragment (left panel). Fragment lengths specify the base pair at the target site query position (ranging from 2200 base pairs for ‘A’ sites to 1320 base pairs for ‘T’ sites; right panel). (B) Pools of four substrates were cleaved in a single tube digest prior to separating substrates and cleavage products in a single lane of an agarose gel. Cleavage of substrate molecules at the centrally located mCreI target site generates two equal length cleavage products and a ‘bar code’ linking substrate and cleavage band intensities that reports cleavage activity and specificity simultaneously for all four base pair possibilities at single target site base pair positions. Examples of base-specific (central panel) and non-specific or degenerate cleavage patterns (right panel) are depicted.
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Figure 2: In vitro cleavage assay. (A) In vitro ‘bar code’ cleavage assay developed to determine simultaneously cleavage specificity and activity for all four base pair possibilities at single target site positions in a single tube/single gel lane assay. Four primer pairs were used to amplify cleavage substrates from target site plasmids with the target site at the center of the resulting PCR fragment (left panel). Fragment lengths specify the base pair at the target site query position (ranging from 2200 base pairs for ‘A’ sites to 1320 base pairs for ‘T’ sites; right panel). (B) Pools of four substrates were cleaved in a single tube digest prior to separating substrates and cleavage products in a single lane of an agarose gel. Cleavage of substrate molecules at the centrally located mCreI target site generates two equal length cleavage products and a ‘bar code’ linking substrate and cleavage band intensities that reports cleavage activity and specificity simultaneously for all four base pair possibilities at single target site base pair positions. Examples of base-specific (central panel) and non-specific or degenerate cleavage patterns (right panel) are depicted.

Mentions: A competitive in vitro ‘bar code’ cleavage assay was developed to determine mCreI specificity at each target site position for all four base pair possibilities (Figure 2). The target site library consisted of 61 plasmids, each containing a different mCreI target site with one of four base pair possibilities at each target site position from −10 to +10 (Figure 1B) cloned into pDR–GFP-univ, a modified version of the pDR–GFP recombination reporter plasmid (28); (see http://depts.washington.edu/monnatws/plasmids/pDR-GFP%20univ.pdf for details). This provided a common target site library that could be used for both in vitro and in vivo cleavage assays. Sequence-verified target site plasmids were used as substrates to amplify different target sites for in vitro cleavage analyses. In brief, PCR primers were chosen to place each target site position at the center of an amplicon that could be readily distinguished on the basis of amplicon size from fragments containing the other three base pair possibilities at each target site base pair position (Figure 2). PCR reactions contained 200 µM dNTPs (New England Biolabs), 0.4 nM of each primer, 50 ng of the highly purified pDR–GFP-Cre template, 1.5 M betaine and 1 U of Taq thermophilic DNA polymerase in 1× Thermopol buffer (New England Biolabs). Betaine was required for successful amplification, and necessitated that amplifications be performed immediately after addition. PCR fragments were purified (Qiagen PCR Cleanup) and quantitated by UV spectrometry (Nanodrop), then combined to form substrate pools in which all four base pair possibilities at each target site base pair position were present in equimolar amount.Figure 2.


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

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

In vitro cleavage assay. (A) In vitro ‘bar code’ cleavage assay developed to determine simultaneously cleavage specificity and activity for all four base pair possibilities at single target site positions in a single tube/single gel lane assay. Four primer pairs were used to amplify cleavage substrates from target site plasmids with the target site at the center of the resulting PCR fragment (left panel). Fragment lengths specify the base pair at the target site query position (ranging from 2200 base pairs for ‘A’ sites to 1320 base pairs for ‘T’ sites; right panel). (B) Pools of four substrates were cleaved in a single tube digest prior to separating substrates and cleavage products in a single lane of an agarose gel. Cleavage of substrate molecules at the centrally located mCreI target site generates two equal length cleavage products and a ‘bar code’ linking substrate and cleavage band intensities that reports cleavage activity and specificity simultaneously for all four base pair possibilities at single target site base pair positions. Examples of base-specific (central panel) and non-specific or degenerate cleavage patterns (right panel) are depicted.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 2: In vitro cleavage assay. (A) In vitro ‘bar code’ cleavage assay developed to determine simultaneously cleavage specificity and activity for all four base pair possibilities at single target site positions in a single tube/single gel lane assay. Four primer pairs were used to amplify cleavage substrates from target site plasmids with the target site at the center of the resulting PCR fragment (left panel). Fragment lengths specify the base pair at the target site query position (ranging from 2200 base pairs for ‘A’ sites to 1320 base pairs for ‘T’ sites; right panel). (B) Pools of four substrates were cleaved in a single tube digest prior to separating substrates and cleavage products in a single lane of an agarose gel. Cleavage of substrate molecules at the centrally located mCreI target site generates two equal length cleavage products and a ‘bar code’ linking substrate and cleavage band intensities that reports cleavage activity and specificity simultaneously for all four base pair possibilities at single target site base pair positions. Examples of base-specific (central panel) and non-specific or degenerate cleavage patterns (right panel) are depicted.
Mentions: A competitive in vitro ‘bar code’ cleavage assay was developed to determine mCreI specificity at each target site position for all four base pair possibilities (Figure 2). The target site library consisted of 61 plasmids, each containing a different mCreI target site with one of four base pair possibilities at each target site position from −10 to +10 (Figure 1B) cloned into pDR–GFP-univ, a modified version of the pDR–GFP recombination reporter plasmid (28); (see http://depts.washington.edu/monnatws/plasmids/pDR-GFP%20univ.pdf for details). This provided a common target site library that could be used for both in vitro and in vivo cleavage assays. Sequence-verified target site plasmids were used as substrates to amplify different target sites for in vitro cleavage analyses. In brief, PCR primers were chosen to place each target site position at the center of an amplicon that could be readily distinguished on the basis of amplicon size from fragments containing the other three base pair possibilities at each target site base pair position (Figure 2). PCR reactions contained 200 µM dNTPs (New England Biolabs), 0.4 nM of each primer, 50 ng of the highly purified pDR–GFP-Cre template, 1.5 M betaine and 1 U of Taq thermophilic DNA polymerase in 1× Thermopol buffer (New England Biolabs). Betaine was required for successful amplification, and necessitated that amplifications be performed immediately after addition. PCR fragments were purified (Qiagen PCR Cleanup) and quantitated by UV spectrometry (Nanodrop), then combined to form substrate pools in which all four base pair possibilities at each target site base pair position were present in equimolar amount.Figure 2.

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