Chromosomal context and epigenetic mechanisms control the efficacy of genome editing by rare-cutting designer endonucleases. Nucleic Acids Res. Epub Mar 29

CELLECTIS S.A., Paris, France.
Nucleic Acids Research (Impact Factor: 9.11). 03/2012; 40(13):6367-79. DOI: 10.1093/nar/gks268
Source: PubMed


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.

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    • "This assay was previously demonstrated to be suitable to assess the intrinsic nuclease activity of TALE nuclease without being biased by epigenetic modifications or chromatin context. In addition, we have previously found a good correlation between data obtained with the yeast SSA assay, an extrachromosomal SSA assay in CHO-K1 and chromosomal disruption experiments in CHO-KI [25,26]. We thus believed that the yeast model system could serve as an appropriate and representative assay to compare characteristics of different nuclease architectures. "
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    ABSTRACT: Background The past decade has seen the emergence of several molecular tools that render possible modification of cellular functions through accurate and easy addition, removal, or exchange of genomic DNA sequences. Among these technologies, transcription activator-like effectors (TALE) has turned out to be one of the most versatile and incredibly robust platform for generating targeted molecular tools as demonstrated by fusion to various domains such as transcription activator, repressor and nucleases. Results In this study, we generated a novel nuclease architecture based on the transcription activator-like effector scaffold. In contrast to the existing Tail to Tail (TtT) and head to Head (HtH) nuclease architectures based on the symmetrical association of two TALE DNA binding domains fused to the C-terminal (TtT) or N-terminal (HtH) end of FokI, this novel architecture consists of the asymmetrical association of two different engineered TALE DNA binding domains fused to the N- and C-terminal ends of FokI (TALE::FokI and FokI::TALE scaffolds respectively). The characterization of this novel Tail to Head (TtH) architecture in yeast enabled us to demonstrate its nuclease activity and define its optimal target configuration. We further showed that this architecture was able to promote substantial level of targeted mutagenesis at three endogenous loci present in two different mammalian cell lines. Conclusion Our results demonstrated that this novel functional TtH architecture which requires binding to only one DNA strand of a given endogenous locus has the potential to extend the targeting possibility of FokI-based TALE nucleases.
    Full-text · Article · Jul 2014 · BMC Molecular Biology
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    • "One approach to GE is based on the use of sequence-specific nucleases to trigger DNA modifications by generating DNA double-strand breaks at the locus of interest. Currently, the four most frequently used tools in GE to generate targeted DNA cleavage are transcription activator-like effector nucleases (TALENa) [4], CRISPR nuclease complexes [5,6], zinc-finger nucleases (ZFN) [4,7,8] and meganucleases (MN) [9,10]. These proteins have different properties (size, origin etc.), making it possible to match them with specific applications [3,11]. "
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    ABSTRACT: Background Meganucleases are important tools for genome engineering, providing an efficient way to generate DNA double-strand breaks at specific loci of interest. Numerous experimental efforts, ranging from in vivo selection to in silico modeling, have been made to re-engineer meganucleases to target relevant DNA sequences. Results Here we present a novel in silico method for designing custom meganucleases that is based on the use of a machine learning approach. We compared it with existing in silico physical models and high-throughput experimental screening. The machine learning model was used to successfully predict active meganucleases for 53 new DNA targets. Conclusions This new method shows competitive performance compared with state-of-the-art in silico physical models, with up to a fourfold increase in terms of the design success rate. Compared to experimental high-throughput screening methods, it reduces the number of screening experiments needed by a factor of more than 100 without affecting final performance.
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    • "MNs and ZFNs have been used to test the feasibility of activating the NHEJ repair mechanism and of restoring the normal reading frame of a dog microdystrophin gene containing a frame-shift mutation (Chapdelaine et al., 2010; Rousseau et al., 2011). The ability of MNs to induce indels in the dystrophin locus has also been demonstrated through studies aimed at determining the effect of chromatin accessibility on genome editing mediated by MNs (Daboussi et al., 2012). In this study, Daboussi et al designed 37 MNs capable of cleaving different genomic targets. "
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