Duchenne muscular dystrophy (DMD), caused by mutations in the X-linked dystrophin gene (DMD), is characterized by fatal degeneration of striated muscles. Dilated cardiomyopathy is one of the most common lethal features of the disease. We deployed Cpf1, a unique class 2 CRISPR (clustered regularly interspaced short palindromic repeats) effector, to correct DMD mutations in patient-derived induced pluripotent stem cells (iPSCs) and mdx mice, an animal model of DMD. Cpf1-mediated genomic editing of human iPSCs, either by skipping of an out-of-frame DMD exon or by correcting a nonsense mutation, restored dystrophin expression after differentiation to cardiomyocytes and enhanced contractile function. Similarly, pathophysiological hallmarks of muscular dystrophy were corrected in mdx mice following Cpf1-mediated germline editing. These findings are the first to show the efficiency of Cpf1-mediated correction of genetic mutations in human cells and an animal disease model and represent a significant step toward therapeutic translation of gene editing for correction of DMD.

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INTRODUCTION

Duchenne muscular dystrophy (DMD) is an X-linked recessive disease caused by mutations in the gene coding for dystrophin, which is a large cytoskeletal protein essential for the integrity of muscle cell membranes (1). DMD causes progressive muscle weakness, culminating in premature death by the age of 30 years, generally from cardiomyopathy. There is no effective treatment for this disease. Numerous approaches to rescue dystrophin expression in DMD have been attempted, including delivery of truncated dystrophin or utrophin by recombinant adeno-associated virus (AAV) (2, 3) and skipping of mutant exons with antisense oligonucleotides and small molecules (4). However, these approaches cannot correct DMD mutations or permanently restore dystrophin expression.

The CRISPR (clustered regularly interspaced short palindromic repeats) system functions as an adaptive immune system in bacteria and archaea that defends against phage infection (5). In this system, an endonuclease is guided to specific genomic sequences by a single guide RNA (sgRNA), resulting in DNA cutting near a protospacer adjacent motif (PAM) sequence. The CRISPR-Cas (CRISPR-associated proteins) system represents a promising approach for correction of diverse genetic defects (6–12). However, many challenges remain to be addressed. For example, Streptococcus pyogenes Cas9 (SpCas9), currently the most widely used Cas9 endonuclease, has a G-rich PAM requirement (NGG) that excludes genome editing of AT-rich regions (13). Additionally, the large size of SpCas9 reduces the efficiency of packaging and delivery in low-capacity viral vectors, such as AAV vectors. The Cas9 endonuclease from Staphylococcus aureus (SaCas9), although smaller in size than SpCas9, has a PAM sequence (NNGRRT) that is longer and more complex, thus limiting the range of its genomic targets (13). Smaller CRISPR enzymes with greater flexibility in recognition sequence and comparable cutting efficiency would facilitate precision gene editing, especially for translational applications.

Recently, a new RNA-guided endonuclease, named Cpf1 (CRISPR from Prevotella and Francisella 1), was shown to be effective in mammalian genome cleavage (14–18). Cpf1 has several unique features that expand its genome editing potential: (i) Cpf1-mediated cleavage is guided by a single and short crRNA (abbreviated as gRNA), whereas Cas9-mediated cleavage is guided by a hybrid of CRISPR RNA (crRNA) and a long trans-activating crRNA (tracrRNA) (19). (ii) Cpf1 prefers a T-rich PAM at the 5′ end of a protospacer, whereas Cas9 requires a G-rich PAM at the 3′ end of the target sequence. (iii) Cpf1-mediated cleavage produces a sticky end distal to the PAM site, which activates DNA repair machinery, whereas Cas9 cutting generates a blunt end. (iv) Cpf1 also has ribonuclease activity, which can process precursor crRNAs to mature crRNAs (14, 20). Like Cas9, Cpf1 binds to a targeted genomic site and generates a double-stranded break, which is then repaired either by nonhomologous end joining (NHEJ) or by homology-directed repair (HDR) if an exogenous template is provided. Although Cpf1 has been shown to be active in mammalian genome editing (14–17), its potential usefulness for correction of genetic mutations in mammalian cells and animal models of disease has yet to be demonstrated.

Previously, we and others used CRISPR-Cas9 to correct the DMD mutation in mice and human cells (7, 9, 10, 21–23). Here, we show that Cpf1 provides a robust and efficient RNA-guided genome editing system that can be used to permanently correct DMD mutations by different strategies, thereby restoring dystrophin expression and preventing progression of the disease. These findings provide a new approach for the permanent correction of human genetic mutations.