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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.


Specificity analysis of redesigned BinQ variants.Recombination by BinQ-CCR5 L and R, and BinQ-AAVS1 L and R on 20-Bin core sites containing (A) all posssible weak (W: A or T) substitutions within positions 6–4, or the dinucleotide core (±1) substitution GG and (B) all possible two-base combinations within positions 3–2. Recombination was determined by split gene assembly.
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pone.0139123.g008: Specificity analysis of redesigned BinQ variants.Recombination by BinQ-CCR5 L and R, and BinQ-AAVS1 L and R on 20-Bin core sites containing (A) all posssible weak (W: A or T) substitutions within positions 6–4, or the dinucleotide core (±1) substitution GG and (B) all possible two-base combinations within positions 3–2. Recombination was determined by split gene assembly.

Mentions: In order to more fully characterize the activity of each selected recombinase variant, we next evaluated the substrate specificity profile of BinQ-AAVS1 L and R, and BinQ-CCR5 L and R. This was achieved by introducing each possible weak base (A or T) substitution into positions 6–4, and each possible two-base combination into positions 3–2 within the 20-bp core site recognized by each BinQ variant. Compared to the parent clone, both BinQ-AAVS1 L and BinQ-CCR5 L displayed increased specificity for their intended target site, demonstrating low levels of recombination (<0.1%) on substrates containing even a single T substitution anywhere within positions 6–4 (Fig 8A). BinQ-AAVS1 L and BinQ-CCR5 L also exhibited minimal amounts of recombination when tested on core sites containing the dinucleotide core (±1) substitution GG. Similarly, both BinQ-AAVS1 R and BinQ-CCR5 R displayed a 10-fold decrease in recombination on substrates harboring any weak substitutions within positions 6–4 (Fig 8A). For positions 3–2, all evolved variants demonstrated some off-target activity, with substrates containing CA, GA, CT and GT substitutions yielding the highest levels of non-specific recombination (Fig 8B). Additionally, both BinQ-AAVS1 R and BinQ-CCR5 R showed increased off-target recombination for each substrate harboring a weak two-base substitution at positions 3–2. Together, these results demonstrate that enzyme variants capable of specific recombination of target sites from the CCR5 gene and AAVS1 locus can be generated by protein engineering methods.


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

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

Specificity analysis of redesigned BinQ variants.Recombination by BinQ-CCR5 L and R, and BinQ-AAVS1 L and R on 20-Bin core sites containing (A) all posssible weak (W: A or T) substitutions within positions 6–4, or the dinucleotide core (±1) substitution GG and (B) all possible two-base combinations within positions 3–2. Recombination was determined by split gene assembly.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0139123.g008: Specificity analysis of redesigned BinQ variants.Recombination by BinQ-CCR5 L and R, and BinQ-AAVS1 L and R on 20-Bin core sites containing (A) all posssible weak (W: A or T) substitutions within positions 6–4, or the dinucleotide core (±1) substitution GG and (B) all possible two-base combinations within positions 3–2. Recombination was determined by split gene assembly.
Mentions: In order to more fully characterize the activity of each selected recombinase variant, we next evaluated the substrate specificity profile of BinQ-AAVS1 L and R, and BinQ-CCR5 L and R. This was achieved by introducing each possible weak base (A or T) substitution into positions 6–4, and each possible two-base combination into positions 3–2 within the 20-bp core site recognized by each BinQ variant. Compared to the parent clone, both BinQ-AAVS1 L and BinQ-CCR5 L displayed increased specificity for their intended target site, demonstrating low levels of recombination (<0.1%) on substrates containing even a single T substitution anywhere within positions 6–4 (Fig 8A). BinQ-AAVS1 L and BinQ-CCR5 L also exhibited minimal amounts of recombination when tested on core sites containing the dinucleotide core (±1) substitution GG. Similarly, both BinQ-AAVS1 R and BinQ-CCR5 R displayed a 10-fold decrease in recombination on substrates harboring any weak substitutions within positions 6–4 (Fig 8A). For positions 3–2, all evolved variants demonstrated some off-target activity, with substrates containing CA, GA, CT and GT substitutions yielding the highest levels of non-specific recombination (Fig 8B). Additionally, both BinQ-AAVS1 R and BinQ-CCR5 R showed increased off-target recombination for each substrate harboring a weak two-base substitution at positions 3–2. Together, these results demonstrate that enzyme variants capable of specific recombination of target sites from the CCR5 gene and AAVS1 locus can be generated by protein engineering methods.

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.