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Redesigning Recombinase Specificity for Safe Harbor Sites in the Human Genome.

Wallen MC, Gaj T, Barbas CF - PLoS ONE (2015)

Bottom Line: Engineered zinc-finger and TAL effector recombinases, in particular, are two classes of SSRs composed of custom-designed DNA-binding domains fused to a catalytic domain derived from the resolvase/invertase family of serine recombinases.While TAL effector and zinc-finger proteins can be assembled to recognize a wide range of possible DNA sequences, recombinase catalytic specificity has been constrained by inherent base requirements present within each enzyme.Taken together, these findings demonstrate that complementing functional characterization with protein engineering is a potentially powerful approach for generating recombinases with expanded targeting capabilities.

View Article: PubMed Central - PubMed

Affiliation: The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA, 92037, United States of America; Department of Chemistry, The Scripps Research Institute, La Jolla, CA, 92037, United States of America; Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, CA, 92037, United States of America.

ABSTRACT
Site-specific recombinases (SSRs) are valuable tools for genetic engineering due to their ability to manipulate DNA in a highly specific manner. Engineered zinc-finger and TAL effector recombinases, in particular, are two classes of SSRs composed of custom-designed DNA-binding domains fused to a catalytic domain derived from the resolvase/invertase family of serine recombinases. While TAL effector and zinc-finger proteins can be assembled to recognize a wide range of possible DNA sequences, recombinase catalytic specificity has been constrained by inherent base requirements present within each enzyme. In order to further expand the targeted recombinase repertoire, we used a genetic screen to isolate enhanced mutants of the Bin and Tn21 recombinases that recognize target sites outside the scope of other engineered recombinases. We determined the specific base requirements for recombination by these enzymes and demonstrate their potential for genome engineering by selecting for variants capable of specifically recombining target sites present in the human CCR5 gene and the AAVS1 safe harbor locus. Taken together, these findings demonstrate that complementing functional characterization with protein engineering is a potentially powerful approach for generating recombinases with expanded targeting capabilities.

No MeSH data available.


Recombination efficiency of selected Bin and Tn21 catalytic domain variants.The activity of selected (A) Bin and (B) Tn21 catalytic domains was evaluated against a panel of cognate and non-cognate target sites. Red highlighted variants were selected for further analysis. Recombination was determined by split gene assembly.
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pone.0139123.g004: Recombination efficiency of selected Bin and Tn21 catalytic domain variants.The activity of selected (A) Bin and (B) Tn21 catalytic domains was evaluated against a panel of cognate and non-cognate target sites. Red highlighted variants were selected for further analysis. Recombination was determined by split gene assembly.

Mentions: We next used split gene reassembly to measure the ability of individually selected Bin and Tn21 variants to recombine both cognate core sites (i.e., 20-Bin and 20-Tn21) and non-cognate (i.e., 20B, 20G, 20S and 20T) 20-bp core sites (Table 1). Among all analyzed Bin clones, BinQ (H34R, N78S, F87I, D97G and K143E) displayed the highest level of specificity for its intended DNA target (Fig 4A). In contrast to past studies, subtracting any single selected BinQ mutation dramatically reduced enzyme specificity and/or efficiency, indicating that the


Redesigning Recombinase Specificity for Safe Harbor Sites in the Human Genome.

Wallen MC, Gaj T, Barbas CF - PLoS ONE (2015)

Recombination efficiency of selected Bin and Tn21 catalytic domain variants.The activity of selected (A) Bin and (B) Tn21 catalytic domains was evaluated against a panel of cognate and non-cognate target sites. Red highlighted variants were selected for further analysis. Recombination was determined by split gene assembly.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4587366&req=5

pone.0139123.g004: Recombination efficiency of selected Bin and Tn21 catalytic domain variants.The activity of selected (A) Bin and (B) Tn21 catalytic domains was evaluated against a panel of cognate and non-cognate target sites. Red highlighted variants were selected for further analysis. Recombination was determined by split gene assembly.
Mentions: We next used split gene reassembly to measure the ability of individually selected Bin and Tn21 variants to recombine both cognate core sites (i.e., 20-Bin and 20-Tn21) and non-cognate (i.e., 20B, 20G, 20S and 20T) 20-bp core sites (Table 1). Among all analyzed Bin clones, BinQ (H34R, N78S, F87I, D97G and K143E) displayed the highest level of specificity for its intended DNA target (Fig 4A). In contrast to past studies, subtracting any single selected BinQ mutation dramatically reduced enzyme specificity and/or efficiency, indicating that the

Bottom Line: Engineered zinc-finger and TAL effector recombinases, in particular, are two classes of SSRs composed of custom-designed DNA-binding domains fused to a catalytic domain derived from the resolvase/invertase family of serine recombinases.While TAL effector and zinc-finger proteins can be assembled to recognize a wide range of possible DNA sequences, recombinase catalytic specificity has been constrained by inherent base requirements present within each enzyme.Taken together, these findings demonstrate that complementing functional characterization with protein engineering is a potentially powerful approach for generating recombinases with expanded targeting capabilities.

View Article: PubMed Central - PubMed

Affiliation: The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA, 92037, United States of America; Department of Chemistry, The Scripps Research Institute, La Jolla, CA, 92037, United States of America; Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, CA, 92037, United States of America.

ABSTRACT
Site-specific recombinases (SSRs) are valuable tools for genetic engineering due to their ability to manipulate DNA in a highly specific manner. Engineered zinc-finger and TAL effector recombinases, in particular, are two classes of SSRs composed of custom-designed DNA-binding domains fused to a catalytic domain derived from the resolvase/invertase family of serine recombinases. While TAL effector and zinc-finger proteins can be assembled to recognize a wide range of possible DNA sequences, recombinase catalytic specificity has been constrained by inherent base requirements present within each enzyme. In order to further expand the targeted recombinase repertoire, we used a genetic screen to isolate enhanced mutants of the Bin and Tn21 recombinases that recognize target sites outside the scope of other engineered recombinases. We determined the specific base requirements for recombination by these enzymes and demonstrate their potential for genome engineering by selecting for variants capable of specifically recombining target sites present in the human CCR5 gene and the AAVS1 safe harbor locus. Taken together, these findings demonstrate that complementing functional characterization with protein engineering is a potentially powerful approach for generating recombinases with expanded targeting capabilities.

No MeSH data available.