Reading Frame Correction by Targeted Genome Editing Restores Dystrophin Expression in Cells From Duchenne Muscular Dystrophy Patients

Ousterout, David G.; Pérez-Piñera, Pablo; Thakore, Pratiksha I.; Kabadi, Ami M.; Brown, Matthew T.; Qin, Xiao; Fédrigo, Olivier; Mouly, Vincent; Tremblay, Jacques P.; Gersbach, Charles A.

doi:10.1038/mt.2013.111

Cited by 164 publications

(140 citation statements)

References 50 publications

Supporting

Mentioning

136

Contrasting

Unclassified

Order By: Relevance

“…PCR enriched libraries were subjected to Illumina HiSeq 2000 at Bio Medical Laboratories. Exome sequence data were analyzed essentially as described (Ousterout et al 2013). Raw reads in FASTQ format were aligned to the NCBI36/Hg18 reference genome using BWA-0.5.9.…”

Section: Exome Sequencingmentioning

confidence: 99%

Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases

et al. 2013

View full text Add to dashboard Cite

RNA-guided endonucleases (RGENs), derived from the prokaryotic adaptive immune system known as CRISPR/Cas, enable targeted genome engineering in cells and organisms. RGENs are ribonucleoproteins that consist of guide RNA and Cas9, a protein component originated from Streptococcus pyogenes. These enzymes cleave chromosomal DNA, whose sequence is complementary, to guide RNA in a targeted manner, producing site-specific DNA double-strand breaks (DSBs), the repair of which gives rise to targeted genome modifications. Despite broad interest in RGEN-mediated genome editing, these nucleases are limited by off-target mutations and unwanted chromosomal translocations associated with off-target DNA cleavages. Here, we show that off-target effects of RGENs can be reduced below the detection limits of deep sequencing by choosing unique target sequences in the genome and modifying both guide RNA and Cas9. We found that both the composition and structure of guide RNA can affect RGEN activities in cells to reduce off-target effects. RGENs efficiently discriminated on-target sites from off-target sites that differ by two bases. Furthermore, exome sequencing analysis showed that no off-target mutations were induced by two RGENs in four clonal populations of mutant cells. In addition, paired Cas9 nickases, composed of D10A Cas9 and guide RNA, which generate two single-strand breaks (SSBs) or nicks on different DNA strands, were highly specific in human cells, avoiding off-target mutations without sacrificing genome-editing efficiency. Interestingly, paired nickases induced chromosomal deletions in a targeted manner without causing unwanted translocations. Our results highlight the importance of choosing unique target sequences and optimizing guide RNA and Cas9 to avoid or reduce RGEN-induced off-target mutations.

show abstract

Section: Exome Sequencingmentioning

confidence: 99%

Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases

et al. 2013

View full text Add to dashboard Cite

show abstract

“…79,131 Besides answering these basic gene therapy questions, the cDMD model will also be essential to determine whether strategies that are shown to protect mouse muscle can or cannot treat muscular dystrophy in large mammals. New technologies that have (such as the use of the dual-AAV system to express a 6-8 kb minidystrophin gene) [145][146][147] or have not (such as the use of nuclease to correct dystrophin gene mutation in vivo) [148][149][150] been tested in mice may ultimately require corroboration in the canine model.…”

Section: Summary and Perspectivesmentioning

confidence: 99%

Duchenne muscular dystrophy gene therapy in the canine model

Duan¹

2015

Human Gene Therapy Clinical Development

View full text Add to dashboard Cite

Duchenne muscular dystrophy (DMD) is an X-linked lethal muscle disease caused by dystrophin deficiency. Gene therapy has significantly improved the outcome of dystrophin-deficient mice. Yet, clinical translation has not resulted in the expected benefits in human patients. This translational gap is largely because of the insufficient modeling of DMD in mice. Specifically, mice lacking dystrophin show minimum dystrophic symptoms, and they do not respond to the gene therapy vector in the same way as human patients do. Further, the size of a mouse is hundredfolds smaller than a boy, making it impossible to scale-up gene therapy in a mouse model. None of these limitations exist in the canine DMD (cDMD) model. For this reason, cDMD dogs have been considered a highly valuable platform to test experimental DMD gene therapy. Over the last three decades, a variety of gene therapy approaches have been evaluated in cDMD dogs using a number of nonviral and viral vectors. These studies have provided critical insight for the development of an effective gene therapy protocol in human patients. This review discusses the history, current status, and future directions of the DMD gene therapy in the canine model.

show abstract

“…It could be shown that dsDNA breaks caused by TALENs induce homologous recombination in the presence of homologous sequences contained in the respective donor DNA [64][65][66]. This mechanism can be used to correct mutations associated with genetic diseases by transducing a DNA sequence homologous to the target sequence along with the TALEN encoding sequences.…”

Section: Future Viral Vectors For Delivery Of Talensmentioning

confidence: 99%

Progress and Problems with Viral Vectors for Delivery of Talens

Bergmann¹

2014

J Mol Genet Med

View full text Add to dashboard Cite

Transcription Activator-Like Effector Nucleases (TALENS) and Their Mode of ActionFor sequence-specific genome engineering and its biotechnological and medical applications various systems were tested. Most recent tools include designer nucleases and the bacteria derived clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system using short RNA to induce precise cleavage at endogenous genomic loci [1,2]. Designer nucleases are mainly represented by zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) [3][4][5][6][7], combining features of a customized DNA binding motif for sequence-specific DNA binding domain and a nuclease for introduction of doubled-strand DNA (dsDNA) breaks at the target site.TALENs are a promising new class of designer nucleases that can be specifically designed to bind DNA sequences of interest and to introduce dsDNA breaks. They are chimeric proteins consisting of a N-terminal nuclear localization signal, a central DNA binding domain and a C-terminal FokI nuclease domain. The DNA binding domain originates from transcription activator like effectors (TALEs) of the bacterial plant pathogen Xanthomonas, used by this bacterium to alter the expression of several host genes [8]. Its main characteristic is a central repeat region consisting of a variable number of incomplete tandem repeats that are usually comprised of 33-35 amino acids (aa) that are identical except for the hypervariable repeat-variable diresidue (RVD) at a positions 12 and 13 [6]. With some degree of degeneracy, the RVD of each repeat is specific for binding a corresponding nucleotide in their contiguous target DNA sequence [6,8]. The C-terminal FokI domain provides non-specific endonuclease activity and similar to ZFNs, binding of the TALE domains upstream and downstream of the respective DNA target and subsequent dimerization at the FokI nuclease domain lead to dsDNA breaks. It is of note that the spacer between the TALEN DNA binding sites needs to be considered when designing novel TALENs [6,7]. After binding, dsDNA breaks are introduced which can than activate cellular pathways either leading to homologous recombination in the presence of the respective homologous donor AbstractIt has long been envisaged that gene disruption or gene correction in affected target cells can be efficiently conducted in vitro and in vivo and over the recent years several tools for achieving this goal were developed. Designer nucleases such as zinc finger nucleases (ZFNs) were extensively explored and more recently transcription activator-like effector nucleases (TALENs) were introduced for sequence-specific genome engineering in the mammalian genome. ZFNs and TALENs are fusion proteins containing a customized DNA-binding motif for sequence-specific DNA binding linked to a nuclease for introduction of double-stranded DNA breaks. Both systems were explored in mammalian cells using non-viral and viral delivery methods. Herein, we will provide a state-ofthe-art overview of available virus-base...

show abstract

Reading Frame Correction by Targeted Genome Editing Restores Dystrophin Expression in Cells From Duchenne Muscular Dystrophy Patients

Cited by 164 publications

References 50 publications

Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases

Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases

Duchenne muscular dystrophy gene therapy in the canine model

Progress and Problems with Viral Vectors for Delivery of Talens

Contact Info

Product

Resources

About