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

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

ABSTRACT 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.

Download full-text


Available from: George Dickson, Jul 17, 2015
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The progressive loss of muscle mass characteristic of many muscular dystrophies impairs the efficacy of most of the gene and molecular therapies currently being pursued for the treatment of those disorders. It is becoming increasingly evident that a therapeutic application, to be effective, needs to target not only mature myofibers, but also muscle progenitors cells or muscle stem cells able to form new muscle tissue and to restore myofibers lost as the result of the diseases or during normal homeostasis so as to guarantee effective and lost lasting effects. Correction of the genetic defect using oligodeoxynucleotides (ODNs) or engineered nucleases holds great potential for the treatment of many of the musculoskeletal disorders. The encouraging results obtained by studying in vitro systems and model organisms have set the groundwork for what is likely to become an emerging field in the area of molecular and regenerative medicine. Furthermore, the ability to isolate and expand from patients various types of muscle progenitor cells capable of committing to the myogenic lineage provides the opportunity to establish cell lines that can be used for transplantation following ex vivo manipulation and expansion. The purpose of this article is to provide a perspective on approaches aimed at correcting the genetic defect using gene editing strategies and currently under development for the treatment of Duchenne muscular dystrophy (DMD), the most sever of the neuromuscular disorders. Emphasis will be placed on describing the potential of using the patient own stem cell as source of transplantation and the challenges that gene editing technologies face in the field of regenerative biology.
    Frontiers in Physiology 04/2014; 5:148. DOI:10.3389/fphys.2014.00148
  • Source
    • "Epigenetic status is known to affect all DNA-metabolism processes including transcription, replication, and repair (Cavalli and Misteli, 2013; Papamichos-Chronakis and Peterson, 2013). The importance of transcriptional activation and epigenetic status for gene-editing efficiency is still largely unknown, but epigenetic modification was recently shown to impact DSBR pathway choice (Daboussi et al., 2012; Kuhar et al., 2014; Valton et al., 2012a, 2012b; van Rensburg et al., 2013). The SMRT DNA sequencing strategy could be used to further study how chromatin status influences DSBR pathway choice and gene-editing efficiency by providing analysis in a broader range of cell types in which the chromatin state of the targeted site is known. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Targeted genome editing with engineered nucleases has transformed the ability to introduce precise sequence modifications at almost any site within the genome. A major obstacle to probing the efficiency and consequences of genome editing is that no existing method enables the frequency of different editing events to be simultaneously measured across a cell population at any endogenous genomic locus. We have developed a method for quantifying individual genome-editing outcomes at any site of interest with single-molecule real-time (SMRT) DNA sequencing. We show that this approach can be applied at various loci using multiple engineered nuclease platforms, including transcription-activator-like effector nucleases (TALENs), RNA-guided endonucleases (CRISPR/Cas9), and zinc finger nucleases (ZFNs), and in different cell lines to identify conditions and strategies in which the desired engineering outcome has occurred. This approach offers a technique for studying double-strand break repair, facilitates the evaluation of gene-editing technologies, and permits sensitive quantification of editing outcomes in almost every experimental system used.
    Cell Reports 03/2014; DOI:10.1016/j.celrep.2014.02.040
  • Source
    • "Construction of lentivirus-based MN and targeting matrix plasmids A meganuclease (MN-DMD31), derived from I-CreI and engineered as described previously (Smith et al., 2006; Grizot et al., 2011; Daboussi et al., 2012), has been designed and developed to target intron 44 within the DMD gene, upstream of a mutation hotspot. Single-chain MN constructs of the two variants of DMD31 (3631 and 3633) were subcloned into the MluI/SwaI-digested lentiviral (LV) transfer plasmid (pRRLscSegfpCncs1w), with enhanced green fluorescent protein gene (eGFP) coexpression. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Duchenne muscular dystrophy (DMD) is a severe inherited, muscle wasting disorder caused by mutations in the DMD gene. Gene therapy development for DMD has concentrated on vector-based DMD minigene transfer, cell-based gene therapy using genetically modified adult muscle stem cells or healthy wild-type donor cells, and antisense oligonucleotide-induced exon skipping therapy to restore the reading frame of the mutated DMD gene. This study is an investigation into DMD gene targeting-mediated correction of deletions in human patient myoblasts using a target-specific meganuclease (MN) and a homologous recombination repair matrix. The MN was designed to cleave within DMD intron 44, upstream of a deletion hot-spot, and integration-competent lentiviral vectors expressing the nuclease (LVcMN) were generated. MN western blotting and deep gene sequencing for LVcMN-induced non-homologous end joining indels confirmed efficient MN expression and activity in transduced DMD myoblasts. A homologous repair matrix carrying exons 45-52 (RM45-52) was designed and packaged into integration-deficient lentiviral vectors (IDLVs; LVdRM45-52). Following co-transduction of DMD myoblasts harbouring a deletion of exons 45 to 52 with LVcMN and LVdRM45-52 vectors, targeted knock-in of the RM45-52 region in the correct location in DMD intron 44, and expression of full length, correctly spliced wild-type dystrophin mRNA containing exons 45-52 was observed. This work demonstrates that genome surgery of human DMD gene mutations can be achieved by meganuclease-induced locus-specific genome cleavage and homologous recombination knock-in of deleted exons. The feasibility of human DMD gene repair in patient myoblasts has exciting therapeutic potential.
    Human gene therapy 06/2013; DOI:10.1089/hum.2013.081
Show more