Christophe Perez

Molecular basis of xeroderma pigmentosum group C DNA recognition by engineered meganucleases

Xeroderma pigmentosum is a monogenic disease characterized by hypersensitivity to ultraviolet light. The cells of xeroderma pigmentosum patients are defective in nucleotide excision repair, limiting their capacity to eliminate ultraviolet-induced DNA damage, and resulting in a strong predisposition to develop skin cancers. The use of rare cutting DNA endonucleases—such as homing endonucleases, also known as meganucleases—constitutes one possible strategy for repairing DNA lesions. Homing endonucleases have emerged as highly specific molecular scalpels that recognize and cleave DNA sites, promoting efficient homologous gene targeting through double-strand-break-induced homologous recombination. Here we describe two engineered heterodimeric derivatives of the homing endonuclease I-CreI, produced by a semi-rational approach. These two molecules—Amel3–Amel4 and Ini3–Ini4—cleave DNA from the human XPC gene (xeroderma pigmentosum group C), in vitro and in vivo. Crystal structures of the I-CreI variants complexed with intact and cleaved XPC target DNA suggest that the mechanism of DNA recognition and cleavage by the engineered homing endonucleases is similar to that of the wild-type I-CreI. Furthermore, these derivatives induced high levels of specific gene targeting in mammalian cells while displaying no obvious genotoxicity. Thus, homing endonucleases can be designed to recognize and cleave the DNA sequences of specific genes, opening up new possibilities for genome engineering and gene therapy in xeroderma pigmentosum patients whose illness can be treated ex vivo.

Efficient in toto targeted recombination in mouse liver by meganuclease-induced double-strand break.

Sequence‐specific endonucleases with large recognition sites can cleave DNA in living cells, and, as a consequence, stimulate homologous recombination (HR) up to 10 000‐fold. The recent development of artificial meganucleases with chosen specificities has provided the potential to target any chromosomal locus. Thus, they may represent a universal genome engineering tool and seem to be very promising for acute gene therapy. However, in toto applications depend on the ability to target somatic tissues as well as the proficiency of somatic cells to perform double‐strand break (DSB)‐induced HR.

Chromosomal context and epigenetic mechanisms control the efficacy of genome editing by rare-cutting designer endonucleases

The ability to specifically engineer the genome of living cells at precise locations using rare-cutting designer endonucleases has broad implications for biotechnology and medicine, particularly for functional genomics, transgenics and gene therapy. However, the potential impact of chromosomal context and epigenetics on designer endonuclease-mediated genome editing is poorly understood. To address this question, we conducted a comprehensive analysis on the efficacy of 37 endonucleases derived from the quintessential I-CreI meganuclease that were specifically designed to cleave 39 different genomic targets. The analysis revealed that the efficiency of targeted mutagenesis at a given chromosomal locus is predictive of that of homologous gene targeting. Consequently, a strong genome-wide correlation was apparent between the efficiency of targeted mutagenesis (≤0.1% to ∼6%) with that of homologous gene targeting (≤0.1% to ∼15%). In contrast, the efficiency of targeted mutagenesis or homologous gene targeting at a given chromosomal locus does not correlate with the activity of individual endonucleases on transiently transfected substrates. Finally, we demonstrate that chromatin accessibility modulates the efficacy of rare-cutting endonucleases, accounting for strong position effects. Thus, chromosomal context and epigenetic mechanisms may play a major role in the efficiency rare-cutting endonuclease-induced genome engineering.

Factors affecting double-strand break-induced homologous recombination in mammalian cells

Double-strand break (DSB)-induced homologous recombination (HR) of direct repeats is a powerful means to achieve gene excision, a critical step in genome engineering. In this report we have used an extrachrmosomal reporter system to monitor the impact of different parameters on meganuclease-induced HR in CHO-K1 cells. We found that repeat homology length is critical. Virtually no HR could be detected with a 15-bp duplication, while, with repeats larger than 400 bp, recombination efficiency became less dependent on homology length. The presence of an intervening sequence between the duplications dramatically impairs HR, independent of the cleavage position; by 3 kb of insertion, HR is virtually undetectable. Efficient HR can be restored by positioning cleavage sites at both ends of the intervening sequence, allowing a constant level of excision with up to 10 kb of intervening sequences. Using similar constructs, 2.8-kb inserts could be efficiently removed from several chromosomal loci, illustrating the wide potential of this technology. These results fit current models of direct repeat recombination and identify DSB-induced HR as a powerful tool for gene excision.

High Frequency Targeted Mutagenesis Using Engineered Endonucleases and DNA-End Processing Enzymes

Targeting DNA double-strand breaks is a powerful strategy for gene inactivation applications. Without the use of a repair plasmid, targeted mutagenesis can be achieved through Non-Homologous End joining (NHEJ) pathways. However, many of the DNA breaks produced by engineered nucleases may be subject to precise re-ligation without loss of genetic information and thus are likely to be unproductive. In this study, we combined engineered endonucleases and DNA-end processing enzymes to increase the efficiency of targeted mutagenesis, providing a robust and efficient method to (i) greatly improve targeted mutagenesis frequency up to 30-fold, and; (ii) control the nature of mutagenic events using meganucleases in conjunction with DNA-end processing enzymes in human primary cells.

Identification of Genes Regulating Gene Targeting by a High-Throughput Screening Approach

Homologous gene targeting (HGT) is a precise but inefficient process for genome engineering. Several methods for increasing its efficiency have been developed, including the use of rare cutting endonucleases. However, there is still room for improvement, as even nuclease-induced HGT may vary in efficiency as a function of the nuclease, target site, and cell type considered. We have developed a high-throughput screening assay for the identification of factors stimulating meganuclease-induced HGT. We used this assay to explore a collection of siRNAs targeting 19,121 human genes. At the end of secondary screening, we had identified 64 genes for which knockdown affected nuclease-induced HGT. Two of the strongest candidates were characterized further. We showed that siRNAs directed against the ATF7IP gene, encoding a protein involved in chromatin remodeling, stimulated HGT by a factor of three to eight, at various loci and in different cell types. This method thus led to the identification of a number of genes, the manipulation of which might increase rates of targeted recombination.

964. Combinatorial Process for Engineering Specific Meganucleases with Tailored Specificities toward a Natural Target in the XPC Gene

Xeroderma pigmentosum (XP) is a rare disease transmitted in an autosomal recessive manner Patients have an extreme sensitivity to sunlight and develop serious sunburns with onset of poikilodermia in the light-exposed skin. Skin cancers already appear in childhood, and the disease can also be associated with neurological defects. XP results from defects in the Nucleotide Excision Repair (NER) pathway, a DNA maintenance system which removes UV induced DNA damage such as cyclobutane pyrimidine dimers. XP Patients were assigned to 7 complementation groups (XP-A to XP-G), each complementation group resulting from mutations in a distinct NER gene. There is no treatment, and the majority of patients die before reaching adulthood because of metastases. However, skin engraftment can be made locally, but with the general limitations of grafts. Thus gene and cell therapy represents a huge hope for this kind of disease. Meganucleases are at the basis of a new kind of gene therapy for inherited monogenic disease, based on gene correction instead of gene complementation. These sequence-specific endonucleases can stimulate homologous gene targeting by several orders of magnitude, thereby enabling the correction of mutated genes with significant efficiency. Recently, meganucleases have been used to induce targeted recombination events in mouse hepatocytes with high efficiency, paving the way for therapeutic applications. One of the major challenges is to tailor artificial meganucleases cleaving the gene of interest, while keeping high levels of specificity. We have used a semi-rational approach to produce meganucleases targeting the XPC gene. These novel meganucleases display high levels of activity and specificity. Such results identify modularity of different functional subdomains in the I-CreI DNA-binding scaffold that can be modified without altering the overall structure, and be combined to achieve novel specificities.

417. Meganuclease-Mediated Chromosomal Surgery in Living Animals

Targeted somatic DNA recombination in living animals is at the basis of a new approach in molecular medicine. The last decade has seen the emergence of a new class of DNA engineering tools: the meganucleases. These sequence specific endonucleases with large recognition sites can cleave DNA in living cells and stimulate homologous recombination up to 10,000-fold. The recent development of artificial meganucleases with chosen specificities has provided the potential to target any chromosomal locus. Thus, meganucleases may represent a universal genome surgery tool, with significant potential in therapy. However, in toto applications depend on the ability to target somatic tissues as well as the proficiency of somatic cells to perform DSB-induced homologous recombination (DSBR).