Engineering of Large Numbers of Highly Specific Homing Endonucleases that Induce Recombination on Novel DNA Targets

Arnould, Sylvain; Chames, Patrick; Pérez, C.; Lacroix, Emmanuel; Duclert, Aymeric; Epinat, Jean‐Charles; Stricher, François; Petit, Anne-Sophie; Patin, Amélie; Guillier, Sophie; Rolland, Sandra; Prieto, Jesús; Blanco, Francisco J.; Bravo, Jerónimo; Montoya, Guillermo; Serrano, Luís; Duchâteau, Philippe; Pâques, Frédéric

doi:10.1016/j.jmb.2005.10.065

Cited by 174 publications

(188 citation statements)

References 40 publications

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“…We have used structural data to engineer the specificity of the homodimeric I-CreI protein. Using a statistical approach, we could also infer the role of individual contacts between the protein and the target (10,30). First, we locally engineered subdomains of the I-CreI DNA binding interface to cleave DNA targets differing from the I-CreI target by a few consecutive base pairs.…”

Section: Discussionmentioning

confidence: 99%

“…First, we locally engineered subdomains of the I-CreI DNA binding interface to cleave DNA targets differing from the I-CreI target by a few consecutive base pairs. Then, mutations from locally engineered variants were combined into heterodimeric mutants to cleave chosen targets differing from the I-CreI cleavage site over their entire length (10,30). During the first step, engineering relied essentially on the mutation of residues shown to contact the DNA target (31,32).…”

Section: Discussionmentioning

confidence: 99%

“…A thermodynamic analysis showed a significant destabilization of these mutants, that together with the increase in activity could compromise the enzyme specificity increasing its toxicity. To analyze this issue, the cleavage specificities of these variants and the wild type, were studied in vivo, using a previously described yeast assay that monitors meganuclease-induced recombination at 37°C (10,30). The cleavage of all possible base pair combinations at positions 8, 9 and 10 of the right half (R-10NNN) of the target DNA was assayed (64 substrates in total) (Fig.…”

Section: I-dmoi Versus H-drei Domain Amentioning

confidence: 99%

“…A combinatorial and rational approach (10) has been used for engineering the overall specificity of the homodimeric meganuclease I-CreI to cleave specific sequences, such as the human RAG1 and XPC genes (11,12). However, the resulting variants are heterodimers, consisting of 2 engineered monomers that need to be coexpressed in the targeted cell.…”

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Crystal structure of I-DmoI in complex with its target DNA provides new insights into meganuclease engineering

Marcaida

Prieto

Redondo

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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Homing endonucleases, also known as meganucleases, are sequencespecific enzymes with large DNA recognition sites. These enzymes can be used to induce efficient homologous gene targeting in cells and plants, opening perspectives for genome engineering with applications in a wide series of fields, ranging from biotechnology to gene therapy. Here, we report the crystal structures at 2.0 and 2.1 Å resolution of the I-DmoI meganuclease in complex with its substrate DNA before and after cleavage, providing snapshots of the catalytic process. Our study suggests that I-DmoI requires only 2 cations instead of 3 for DNA cleavage. The structure sheds light onto the basis of DNA binding, indicating key residues responsible for nonpalindromic target DNA recognition. In silico and in vivo analysis of the I-DmoI DNA cleavage specificity suggests that despite the relatively few protein-base contacts, I-DmoI is highly specific when compared with other meganucleases. Our data open the door toward the generation of custom endonucleases for targeted genome engineering using the monomeric I-DmoI scaffold.gene targeting ͉ genetics ͉ protein-DNA interactions ͉ X-ray crystallography M eganucleases are sequence-specific enzymes that recognize large (12-45 bp) DNA target sites. These enzymes are often encoded by introns or inteins behaving as mobile genetic elements. They produce double strand breaks (DSB) that get repaired by homologous recombination with an intron-or intein-containing gene, resulting in the insertion of the intron or intein in that particular site (1). Meganucleases are being used to stimulate homologous gene targeting in the vicinity of their target sequences with the aim of improving current genome engineering approaches, alleviating the risks due to the randomly inserted transgenes (2, 3).The use of meganuclease-induced recombination has long been limited by the repertoire of natural meganucleases. In nature, meganucleases are essentially represented by homing endonucleases, a family of enzymes encoded by mobile genetic elements whose function is to initiate DSB induced recombination events in a process referred to as homing (4). The probability of finding a homing endonuclease cleavage site in a chosen gene is extremely low. Thus, making artificial meganucleases with custom-made substrate specificity is an intense area of research.Sequence homology has been used to classify homing endonucleases into 5 families, the largest one having the conserved LAGLI-DADG sequence motif. Homing endonucleases containing one such motif function as homodimers. In contrast, homing endonucleases containing 2 motifs are single chain proteins (5, 6). Structural information for several members of the LAGLIDADG endonuclease family indicate that these proteins adopt a similar active conformation as homodimers or as monomers with 2 separate domains (7-9). The LAGLIDADG motifs form structurally conserved ␣-helices tightly packed at the center of the interdomain or intermonomer interface. The last acidic residue of this LAGLI-DADG motif part...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: I-dmoi Versus H-drei Domain Amentioning

confidence: 99%

mentioning

confidence: 99%

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Crystal structure of I-DmoI in complex with its target DNA provides new insights into meganuclease engineering

Marcaida

Prieto

Redondo

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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“…29 Engineering highly specific, dedicated DNA endonucleases is the key to a wider usage of this technology. Several groups have developed methods to locally engineer natural MGNs, [30][31][32] and a combinatorial approach allowing for the complete redesign of the MGN DNA-binding interface has been described. 33 Finally, two engineered MGNs cleaving two different human genes, XPC and RAG1, have been described in the literature.…”

Section: Introductionmentioning

confidence: 99%

Meganucleases can restore the reading frame of a mutated dystrophin

et al. 2010

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Mutations in Duchenne muscular dystrophy (DMD) are either inducing a nonsense codon or a frameshift. Meganucleases (MGNs) can be engineered to induce double-strand breaks (DSBs) at specific DNA sequences. These breaks are repaired by homologous recombination or by non-homologous end joining (NHEJ), which results in insertions or deletions (indels) of a few base pairs. To verify whether MGNs could be used to restore the normal reading frame of a dystrophin gene with a frameshift mutation, we inserted in a plasmid coding for the dog m-dystrophin sequences containing a MGN target. The number of base pairs in these inserted sequences changed the reading frame. One of these modified target m-dystrophin plasmids and an appropriate MGN were then transfected in 293FT cells. The MGN induced micro-deletion or micro-insertion in the m-dystrophin that restored dystrophin expression. MGNs also restored m-dystrophin expression in myoblasts in vitro and in muscle fibers in vivo. The mutation of the targeted m-dystrophin was confirmed by PCR amplification followed by digestion with the Surveyor enzyme and by cloning and sequencing of the amplicons. These experiments are thus a proof of principle that MGNs that are adequately engineered to target appropriate sequences in the human dystrophin gene should be able to restore the normal reading frame of that gene in DMD patients with an out-of-frame deletion. New MGNs engineered to target a sequence including or near nonsense mutation could also be used to delete it.

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Meganucleases and Their Biomedical Applications

Molina¹,

Montoya²,

Prieto³

2011

Encyclopedia of Life Sciences

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Homing endonucleases and zinc‐finger nucleases are double‐stranded meganucleases that target large deoxyribonucleic acid (DNA) recognition sites, a feature that means only a few such sites are likely to be present in a mammalian‐sized genome. Several homing endonucleases have been used as templates to engineer tools that cleave DNA sequences other than their original wild‐type targets. Indeed, zinc‐finger nucleases have been designed to recognise specific genes. These custom meganucleases can be used to stimulate double‐strand break homologous recombination in cells, thereby inducing the repair of defective genes with very low toxicity levels. The use of tailored meganucleases opens up new possibilities for gene therapy in patients with monogenic diseases that can be treated ex vivo . This review provides an overview of recent advances in this field. Key Concepts: Meganucleases are double‐stranded DNAses that target large recognition sites. Meganucleases promote efficient gene targeting through double‐strand‐break‐induced homologous recombination. Homologous recombination and nonhomologous end joining are the two pathways that can be used to repair DNA double‐strand breaks. Meganucleases encompass homing endonucleases and zinc‐finger nucleases. Meganucleases are ideal scaffolds to engineer enzymes that accurately cleave DNA and induce recombination. Meganucleases can recognise unique sites of cleavage within a genome, permitting their use for therapeutic purposes in human diseases. X‐ray diffraction of crystallised proteins allows us to understand the relationship between the structure and function of biomolecules.

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Engineering of Large Numbers of Highly Specific Homing Endonucleases that Induce Recombination on Novel DNA Targets

Cited by 174 publications

References 40 publications

Crystal structure of I-DmoI in complex with its target DNA provides new insights into meganuclease engineering

Crystal structure of I-DmoI in complex with its target DNA provides new insights into meganuclease engineering

Meganucleases can restore the reading frame of a mutated dystrophin

Meganucleases and Their Biomedical Applications

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