No abstract

Sequence-specific endonucleases recognizing long target sequences are emerging as powerful tools for genome engineering. These endonucleases could be used to correct deleterious mutations or to inactivate viruses, in a new approach to molecular medicine. However, such applications are highly demanding in terms of safety. Mutations in the human RAG1 gene cause severe combined immunodeficiency (SCID). Using the I-CreI dimeric LAGLIDADG meganuclease as a scaffold, we describe here the engineering of a series of endonucleases cleaving the human RAG1 gene, including obligate heterodimers and single-chain molecules. We show that a novel single-chain design, in which two different monomers are linked to form a single molecule, can induce high levels of recombination while safeguarding more effectively against potential genotoxicity. We provide here the first demonstration that an engineered meganuclease can induce targeted recombination at an endogenous locus in up to 6% of transfected human cells. These properties rank this new generation of endonucleases among the best molecular scissors available for genome surgery strategies, potentially avoiding the deleterious effects of previous gene therapy approaches.

Background: TALE-based technologies are poised to revolutionize the field of biotechnology; however, their sensitivity to cytosine methylation may drastically restrict their ranges of applications. Results: TALE repeat N* proficiently accommodates 5-methylated cytosine. Conclusion: Sensitivity of TALE to cytosine methylation can be overcome by using TALE repeat N*. Significance: Utilization of TALE repeat N* enables broadening the scope of TALE-based technologies.Within the past 2 years, transcription activator-like effector (TALE) DNA binding domains have emerged as the new generation of engineerable platform for production of custom DNA binding domains. However, their recently described sensitivity to cytosine methylation represents a major bottleneck for genome engineering applications. Using a combination of biochemical, structural, and cellular approaches, we were able to identify the molecular basis of such sensitivity and propose a simple, drug-free, and universal method to overcome it.Transcription activator-like effectors (TALEs), 4 a group of bacterial plant pathogen proteins, have recently emerged as new engineerable scaffolds for production of tailored DNA binding domains with chosen specificities (1). Interest in these systems comes from the apparent simple cipher governing DNA recognition by their DNA binding domain (2, 3). The TALE DNA binding domain is composed of multiple TALE repeats that individually recognize one DNA base pair through specific amino acid di-residues (repeat variable di-residues or RVDs). The remarkably high specificity of TALE repeats and the apparent absence of context-dependent effects among repeats in an array allow modular assembly of TALE DNA binding domains able to recognize almost any DNA sequence of interest. Within the past 2 years, engineered TALE DNA binding domains have been fused to transcription activator (dTALEs) (4), repressor (5), or nuclease domains (TALENs) (6) and used to specifically regulate or modify genes of interest (1). Although successfully used in different cellular contexts, engineered TALE DNA binding domains have recently been reported to be affected by the presence of 5-methylated cytosine (5mC) in their endogenous cognate target (7). Often considered as the fifth base, 5mC is found in about 70% of CpG dinucleotides in mammalian and plant somatic/pluripotent cells (8, 9) and has also been reported in 5-cytosine-phosphoadenine, 5-cytosine-phosphothymine, and 5-cytosine-phosphocytosine dinucleotides (10). Moreover, 5mC has been identified in CpG islands embedded in many promoters (11) and, to a higher extent, in proximal exons of several genes (12). These two critical regulatory regions are generally chosen by investigators to knock out genes of therapeutic and biotechnological interest or to modulate their expression using TALE-based technologies. The ubiquity of 5mC in different cell types and genomic kingdoms, its particular localization, and its negative impact on dTALE activity reported in Ref. 7 make this epigenetic modification a major dra...

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.

Recently developed transgenic techniques to explore and exploit the metabolic potential of microalgae present several drawbacks associated with the delivery of exogenous DNA into the cells and its subsequent integration at random sites within the genome. Here, we report a highly efficient multiplex genome-editing method in the diatom Phaeodactylum tricornutum, relying on the biolistic delivery of CRISPR-Cas9 ribonucleoproteins coupled with the identification of two endogenous counter-selectable markers, PtUMPS and PtAPT. First, we demonstrate the functionality of RNP delivery by positively selecting the disruption of each of these genes. Then, we illustrate the potential of the approach for multiplexing by generating double-gene knock-out strains, with 65% to 100% efficiency, using RNPs targeting one of these markers and PtAureo1a, a photoreceptor-encoding gene. Finally, we created triple knock-out strains in one step by delivering six RNP complexes into Phaeodactylum cells. This approach could readily be applied to other hard-to-transfect organisms of biotechnological interest.