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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.


Analysis of hyperactivating mutations in the Bin and Tn21 catalytic domains.(A, B) Frequency and position of the mutations found to hyperactivatate the (A) Bin and (B) Tn21 catalytic domains. Green arrow indicates the catalytic serine residue (C, D) Crystal structures of (C) Gin-M114V (PDB ID: 3UJ3) [66] and (D) Sin-Q115R (PDB ID: 3PKZ) [67], which display homology to the Bin and Tn21 catalytic domains, respectively. Selected mutations present within (C) BinQ and (D) Tn21S are shown as red and blue spheres, respectively.
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pone.0139123.g003: Analysis of hyperactivating mutations in the Bin and Tn21 catalytic domains.(A, B) Frequency and position of the mutations found to hyperactivatate the (A) Bin and (B) Tn21 catalytic domains. Green arrow indicates the catalytic serine residue (C, D) Crystal structures of (C) Gin-M114V (PDB ID: 3UJ3) [66] and (D) Sin-Q115R (PDB ID: 3PKZ) [67], which display homology to the Bin and Tn21 catalytic domains, respectively. Selected mutations present within (C) BinQ and (D) Tn21S are shown as red and blue spheres, respectively.

Mentions: We began by analyzing the activity of the wild-type Bin and Tn21 catalytic domains on minimal crossover sites derived from their native recombination sites. These sites consist of a pseudo-symmetric 20-bp core sequence that contains two inverted 10-bp half-site regions. Specifically, we selected Bin and Tn21 for directed evolution due to their: (i) high sequence similarity to other serine recombinases, and (ii) unique core sites that address “gaps” within the targeted recombinase repertoire. Unlike Gin or any of its evolved variants, the recombination site recognized by Bin contains a TA base combination at positions 3–2, while the crossover site recognized by Tn21 includes G nucleotides at positions 6–4, a region typically restricted to A or T bases for other serine recombinases (Table 1). To measure activity, we used split gene reassembly, a method that directly links recombinase activity to antibiotic resistance in a bacterial host (Fig 2A) [41]. Both Bin and Tn21 demonstrated low levels of recombination (~0.1%) on their intended core sequences. Cross-comparative analysis revealed that hyperactivated variants of the Gin, Tn3, Sin and β catalytic domains also displayed negligible recombination on these substrates, while Sin showed ~10% recombination on the Tn21 core (Fig 2B and 2C). We next used antibiotic selection to identify mutations that enable unrestricted Bin- and Tn21-mediated recombination on their cognate core sequences. Similar approaches have been used to discover hyperactivating mutations for other serine recombinases, including Gin and Hin [53], Tn3 [54], γδ [55], Sin [41, 56] and β [41]. We used error-prone PCR to introduce ~2.5 and ~6 amino acid mutations into the Bin and Tn21 catalytic domains, respectively. We then fused each recombinase library to an unmodified copy of the H1 zinc-finger protein [57], which binds the sequence 5’-GGAGGCGTG-3’ and, in the split gene reassembly selection system, flanks the 20-bp core sequence recognized by the recombinase. After four and five rounds of selection with the Tn21 and Bin libraries, respectively, we observed a >1,000-fold increase in recombination via split gene reassembly (Fig 2C and 2D). We sequenced ~15 clones from each library and observed a number of recurrent mutations that were also commonly found together within singular clones. Among sequenced Bin variants, 65% contained the substitution G103D; 41% contained D97G and M70V/T; and 35% contained H34R (Fig 3A). For Tn21, 68% contained the mutation F14S; 56% contained M63T/V/I; 37% contained F51L/S; and 18% contained H86R/Y (Fig 3B). Hyperactivating mutations have previously been found to cluster near the recombinase E helix and have been proposed to either stabilize the active tetrameric configuration or destabilize the recombinase dimer. Surprisingly, only a few of the resulting Bin and Tn21 mutations were found to reside near the E helix (Fig 3C and 3D), with the majority of the mutations instead located near adjacent loops or the active site.


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

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

Analysis of hyperactivating mutations in the Bin and Tn21 catalytic domains.(A, B) Frequency and position of the mutations found to hyperactivatate the (A) Bin and (B) Tn21 catalytic domains. Green arrow indicates the catalytic serine residue (C, D) Crystal structures of (C) Gin-M114V (PDB ID: 3UJ3) [66] and (D) Sin-Q115R (PDB ID: 3PKZ) [67], which display homology to the Bin and Tn21 catalytic domains, respectively. Selected mutations present within (C) BinQ and (D) Tn21S are shown as red and blue spheres, respectively.
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Related In: Results  -  Collection

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pone.0139123.g003: Analysis of hyperactivating mutations in the Bin and Tn21 catalytic domains.(A, B) Frequency and position of the mutations found to hyperactivatate the (A) Bin and (B) Tn21 catalytic domains. Green arrow indicates the catalytic serine residue (C, D) Crystal structures of (C) Gin-M114V (PDB ID: 3UJ3) [66] and (D) Sin-Q115R (PDB ID: 3PKZ) [67], which display homology to the Bin and Tn21 catalytic domains, respectively. Selected mutations present within (C) BinQ and (D) Tn21S are shown as red and blue spheres, respectively.
Mentions: We began by analyzing the activity of the wild-type Bin and Tn21 catalytic domains on minimal crossover sites derived from their native recombination sites. These sites consist of a pseudo-symmetric 20-bp core sequence that contains two inverted 10-bp half-site regions. Specifically, we selected Bin and Tn21 for directed evolution due to their: (i) high sequence similarity to other serine recombinases, and (ii) unique core sites that address “gaps” within the targeted recombinase repertoire. Unlike Gin or any of its evolved variants, the recombination site recognized by Bin contains a TA base combination at positions 3–2, while the crossover site recognized by Tn21 includes G nucleotides at positions 6–4, a region typically restricted to A or T bases for other serine recombinases (Table 1). To measure activity, we used split gene reassembly, a method that directly links recombinase activity to antibiotic resistance in a bacterial host (Fig 2A) [41]. Both Bin and Tn21 demonstrated low levels of recombination (~0.1%) on their intended core sequences. Cross-comparative analysis revealed that hyperactivated variants of the Gin, Tn3, Sin and β catalytic domains also displayed negligible recombination on these substrates, while Sin showed ~10% recombination on the Tn21 core (Fig 2B and 2C). We next used antibiotic selection to identify mutations that enable unrestricted Bin- and Tn21-mediated recombination on their cognate core sequences. Similar approaches have been used to discover hyperactivating mutations for other serine recombinases, including Gin and Hin [53], Tn3 [54], γδ [55], Sin [41, 56] and β [41]. We used error-prone PCR to introduce ~2.5 and ~6 amino acid mutations into the Bin and Tn21 catalytic domains, respectively. We then fused each recombinase library to an unmodified copy of the H1 zinc-finger protein [57], which binds the sequence 5’-GGAGGCGTG-3’ and, in the split gene reassembly selection system, flanks the 20-bp core sequence recognized by the recombinase. After four and five rounds of selection with the Tn21 and Bin libraries, respectively, we observed a >1,000-fold increase in recombination via split gene reassembly (Fig 2C and 2D). We sequenced ~15 clones from each library and observed a number of recurrent mutations that were also commonly found together within singular clones. Among sequenced Bin variants, 65% contained the substitution G103D; 41% contained D97G and M70V/T; and 35% contained H34R (Fig 3A). For Tn21, 68% contained the mutation F14S; 56% contained M63T/V/I; 37% contained F51L/S; and 18% contained H86R/Y (Fig 3B). Hyperactivating mutations have previously been found to cluster near the recombinase E helix and have been proposed to either stabilize the active tetrameric configuration or destabilize the recombinase dimer. Surprisingly, only a few of the resulting Bin and Tn21 mutations were found to reside near the E helix (Fig 3C and 3D), with the majority of the mutations instead located near adjacent loops or the active site.

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.