br Experimental Procedures br Author Contributions br

Experimental Procedures
Author Contributions
Acknowledgments
Introduction Duchenne muscular dystrophy (DMD) is a severe muscular degenerative disease caused by loss-of-function mutations in the dystrophin gene located on the X chromosome. The dystrophin gene consists of 79 exons, and disruption of the protein reading frame by small deletions, exon duplications, or loss of exons leads to DMD (Pichavant et al., 2011). The large size of the dystrophin gene hampers the delivery of therapeutic cDNA for gene augmentation. Therefore, the delivery of truncated microdystrophin or microutrophin by an adeno-associated viral (AAV) vector (Okada and Takeda, 2013), lentiviral vector (Pichavant et al., 2011), or Sleeping Beauty transposon (Filareto et al., 2013) has been investigated for DMD gene therapy. However, restoration of the full-length dystrophin protein remains challenging. An exon-skipping approach that modulates mRNA splicing patterns using antisense oligonucleotides (Aartsma-Rus et al., 2009) has shown promising results in preclinical studies, but the effects are only transient. Genomic correction using programmable nucleases is an ideal approach that can correct the mutated dystrophin gene. The development of programmable nucleases has provided a powerful tool for modifying target genome sequences. In particular, the transcription activator-like effector nuclease (TALEN) (Hockemeyer et al., 2011) and the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR associated 9 (Cas9) endonuclease systems (Cong et al., 2013; Mali et al., 2013) provide greater flexibility than meganucleases or zinc-finger nucleases (ZFNs) with regard to selecting the target regions of interest (Li et al., 2014). Several studies have demonstrated the effectiveness of TALENs (Hockemeyer et al., 2011; Ding et al., 2013a) and CRISPR (Ding et al., 2013b; Mali et al., 2013) in human induced pluripotent stem Pirfenidone cost (iPSCs) for reporter knockin, gene knockout, and gene correction. In fact, corrections of disease mutation by nucleases in iPSCs have been reported for several diseases, including α1-antitrypsin deficiency (Choi et al., 2013), epidermolysis bullosa (Osborn et al., 2013), β-thalassemia (Ma et al., 2013), AIDS (Ye et al., 2014), and Niemann-Pick Type C (Maetzel et al., 2014). Before the TALEN and CRISPR systems can reach clinical application, however, target specificity must be improved, as high off-target mutagenesis rates in human cells have been reported (Fu et al., 2013; Hsu et al., 2013; Lin et al., 2014), although some reports have shown otherwise (Smith et al., 2014; Suzuki et al., 2014; Veres et al., 2014). Since target specificity depends on the design of the target site, the properties of the DNA-binding domain, and the epigenetic status of the targeting site, the risk of off-target mutagenesis should be examined with respect to each targeting nuclease in a therapeutic setting. Immortalized myoblasts have been used for restoration of the dystrophin protein mediated by meganucleases (Rousseau et al., 2011; Popplewell et al., 2013), ZFNs (Rousseau et al., 2011), or TALENs (Ousterout et al., 2013). However, although primary myoblasts can be derived from patients, their clonal expansion requires transformation by oncogenes such as hTERT. In contrast, iPSCs (Takahashi et al., 2007) can be isolated from patients directly and still maintain pluripotency and an unlimited self-renewal capacity. Accordingly, when conjugated with made-to-order genetic correction technologies, human iPSCs derived from a patient with a genetic disorder (Park et al., 2008; Hotta et al., 2009) might be applicable to autologous transplantation as ex vivo gene therapy (Sebastiano et al., 2011; Soldner et al., 2011; Zou et al., 2011a, 2011b).
Results
Discussion Regarding the nuclease specificity, both TALENs and CRISPR-sgRNA can bind to DNA despite a few base mismatches (Hsu et al., 2013). Therefore, it is critical to target a unique region in the genome with a minimal number of off-targets, as otherwise multiple targets may be cleaved. Several web-based programs can be used to search for off-target sites with a given target sequence region (e.g., CRISPR Design Tool [Hsu et al., 2013], Cas-OFFinder [Bae et al., 2014b], and E-CRISP [Heigwer et al., 2014]). However, these programs provide the predicted number of off-target sites only within a small region (typically ∼500 bp) at any given time. Our unique k-mer approach allows the visualization of targetable regions in the entire genome, so users can select the targetable region(s) before checking the number of off-targets with other programs.