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Expanding LAGLIDADG endonuclease scaffold diversity by rapidly surveying evolutionary sequence space.

Jacoby K, Metzger M, Shen BW, Certo MT, Jarjour J, Stoddard BL, Scharenberg AM - Nucleic Acids Res. (2012)

Bottom Line: One strategy to address such limitations is to identify close homologs of existing LHEs possessing superior biophysical or catalytic properties.Of these enzymes, I-HjeMI demonstrated the greatest activity in vivo and was readily crystallizable, allowing a comparative structural analysis.Taken together, our results suggest that even highly homologous LHEs offer a readily accessible resource of related scaffolds that display diverse biochemical properties for biotechnological applications.

View Article: PubMed Central - PubMed

Affiliation: Program in Molecular and Cellular Biology, University of Washington, Box 357275, Seattle, WA 98195, USA.

ABSTRACT
LAGLIDADG homing endonucleases (LHEs) are a family of highly specific DNA endonucleases capable of recognizing target sequences ≈ 20 bp in length, thus drawing intense interest for their potential academic, biotechnological and clinical applications. Methods for rational design of LHEs to cleave desired target sites are presently limited by a small number of high-quality native LHEs to serve as scaffolds for protein engineering-many are unsatisfactory for gene targeting applications. One strategy to address such limitations is to identify close homologs of existing LHEs possessing superior biophysical or catalytic properties. To test this concept, we searched public sequence databases to identify putative LHE open reading frames homologous to the LHE I-AniI and used a DNA binding and cleavage assay using yeast surface display to rapidly survey a subset of the predicted proteins. These proteins exhibited a range of capacities for surface expression and also displayed locally altered binding and cleavage specificities with a range of in vivo cleavage activities. Of these enzymes, I-HjeMI demonstrated the greatest activity in vivo and was readily crystallizable, allowing a comparative structural analysis. Taken together, our results suggest that even highly homologous LHEs offer a readily accessible resource of related scaffolds that display diverse biochemical properties for biotechnological applications.

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LHE functionality in vivo. (A) Nuclease-expression histogram. Number of cells (Y-axis) of a given mTagBFP fluorescence (X-axis) are shown to be uniform for all transfected cells (solid line) and are compared with an untransfected control (dashed line). Gates used for comparison of expressing and non-expressing populations in panel (B) are shown. (B) Mutagenic NHEJ and HR repair events are shown for each nuclease-expressing population (black) compared with the non-expressing (gray). NHEJ events are mCherry(+) (Y-axis) and HR events are GFP(+) (X-axis). (C) As each mCherry(+) cell represents approximately one-third of the actual mutagenic NHEJ events (18) (Supplementary Figure S1d), a corrected value is plotted for NHEJ events, calculated by multiplying the number of mCherry(+) cells by three. Cells with converted loci, by event type, are shown as a percentage of the total expressing population.
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gkr1303-F4: LHE functionality in vivo. (A) Nuclease-expression histogram. Number of cells (Y-axis) of a given mTagBFP fluorescence (X-axis) are shown to be uniform for all transfected cells (solid line) and are compared with an untransfected control (dashed line). Gates used for comparison of expressing and non-expressing populations in panel (B) are shown. (B) Mutagenic NHEJ and HR repair events are shown for each nuclease-expressing population (black) compared with the non-expressing (gray). NHEJ events are mCherry(+) (Y-axis) and HR events are GFP(+) (X-axis). (C) As each mCherry(+) cell represents approximately one-third of the actual mutagenic NHEJ events (18) (Supplementary Figure S1d), a corrected value is plotted for NHEJ events, calculated by multiplying the number of mCherry(+) cells by three. Cells with converted loci, by event type, are shown as a percentage of the total expressing population.

Mentions: To implement the assay, polyclonal cell lines were generated which harbored integrated single copies of reporters possessing each respective enzymes’ target site. Next, each of these cell lines were transfected with equal amounts of a donor template plasmid which also drives expression of the respective homing endonuclease. This resulted in similar distributions and sums of nuclease expression and repair template copy number, as assessed by the expression (fluorescence) of a monomeric blue fluorescent protein, mTagBFP (Figure 4A). I-AchMI exhibited little to no in vivo activity, consistent with its poor performance in the yeast tethered flow cleavage assay—this may reflect either an actual reduced catalytic efficiency, or that an impaired protein folding and/or thermal stability limits accumulation of active enzyme in cells cultured at 37°C. For these reasons, I-AchMI should be considered a compromised engineering scaffold for in vivo applications.Figure 4.


Expanding LAGLIDADG endonuclease scaffold diversity by rapidly surveying evolutionary sequence space.

Jacoby K, Metzger M, Shen BW, Certo MT, Jarjour J, Stoddard BL, Scharenberg AM - Nucleic Acids Res. (2012)

LHE functionality in vivo. (A) Nuclease-expression histogram. Number of cells (Y-axis) of a given mTagBFP fluorescence (X-axis) are shown to be uniform for all transfected cells (solid line) and are compared with an untransfected control (dashed line). Gates used for comparison of expressing and non-expressing populations in panel (B) are shown. (B) Mutagenic NHEJ and HR repair events are shown for each nuclease-expressing population (black) compared with the non-expressing (gray). NHEJ events are mCherry(+) (Y-axis) and HR events are GFP(+) (X-axis). (C) As each mCherry(+) cell represents approximately one-third of the actual mutagenic NHEJ events (18) (Supplementary Figure S1d), a corrected value is plotted for NHEJ events, calculated by multiplying the number of mCherry(+) cells by three. Cells with converted loci, by event type, are shown as a percentage of the total expressing population.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3367166&req=5

gkr1303-F4: LHE functionality in vivo. (A) Nuclease-expression histogram. Number of cells (Y-axis) of a given mTagBFP fluorescence (X-axis) are shown to be uniform for all transfected cells (solid line) and are compared with an untransfected control (dashed line). Gates used for comparison of expressing and non-expressing populations in panel (B) are shown. (B) Mutagenic NHEJ and HR repair events are shown for each nuclease-expressing population (black) compared with the non-expressing (gray). NHEJ events are mCherry(+) (Y-axis) and HR events are GFP(+) (X-axis). (C) As each mCherry(+) cell represents approximately one-third of the actual mutagenic NHEJ events (18) (Supplementary Figure S1d), a corrected value is plotted for NHEJ events, calculated by multiplying the number of mCherry(+) cells by three. Cells with converted loci, by event type, are shown as a percentage of the total expressing population.
Mentions: To implement the assay, polyclonal cell lines were generated which harbored integrated single copies of reporters possessing each respective enzymes’ target site. Next, each of these cell lines were transfected with equal amounts of a donor template plasmid which also drives expression of the respective homing endonuclease. This resulted in similar distributions and sums of nuclease expression and repair template copy number, as assessed by the expression (fluorescence) of a monomeric blue fluorescent protein, mTagBFP (Figure 4A). I-AchMI exhibited little to no in vivo activity, consistent with its poor performance in the yeast tethered flow cleavage assay—this may reflect either an actual reduced catalytic efficiency, or that an impaired protein folding and/or thermal stability limits accumulation of active enzyme in cells cultured at 37°C. For these reasons, I-AchMI should be considered a compromised engineering scaffold for in vivo applications.Figure 4.

Bottom Line: One strategy to address such limitations is to identify close homologs of existing LHEs possessing superior biophysical or catalytic properties.Of these enzymes, I-HjeMI demonstrated the greatest activity in vivo and was readily crystallizable, allowing a comparative structural analysis.Taken together, our results suggest that even highly homologous LHEs offer a readily accessible resource of related scaffolds that display diverse biochemical properties for biotechnological applications.

View Article: PubMed Central - PubMed

Affiliation: Program in Molecular and Cellular Biology, University of Washington, Box 357275, Seattle, WA 98195, USA.

ABSTRACT
LAGLIDADG homing endonucleases (LHEs) are a family of highly specific DNA endonucleases capable of recognizing target sequences ≈ 20 bp in length, thus drawing intense interest for their potential academic, biotechnological and clinical applications. Methods for rational design of LHEs to cleave desired target sites are presently limited by a small number of high-quality native LHEs to serve as scaffolds for protein engineering-many are unsatisfactory for gene targeting applications. One strategy to address such limitations is to identify close homologs of existing LHEs possessing superior biophysical or catalytic properties. To test this concept, we searched public sequence databases to identify putative LHE open reading frames homologous to the LHE I-AniI and used a DNA binding and cleavage assay using yeast surface display to rapidly survey a subset of the predicted proteins. These proteins exhibited a range of capacities for surface expression and also displayed locally altered binding and cleavage specificities with a range of in vivo cleavage activities. Of these enzymes, I-HjeMI demonstrated the greatest activity in vivo and was readily crystallizable, allowing a comparative structural analysis. Taken together, our results suggest that even highly homologous LHEs offer a readily accessible resource of related scaffolds that display diverse biochemical properties for biotechnological applications.

Show MeSH