Functional disruption of the dystrophin gene in rhesus monkey using CRISPR/Cas9

Abstract

CRISPR/Cas9 has been used to genetically modify genomes in a variety of species, including non-human primates. Unfortunately, this new technology does cause mosaic mutations, and we do not yet know whether such mutations can functionally disrupt the targeted gene or cause the pathology seen in human disease. Addressing these issues is necessary if we are to generate large animal models of human diseases using CRISPR/Cas9. Here we used CRISPR/Cas9 to target the monkey dystrophin gene to create mutations that lead to Duchenne muscular dystrophy (DMD), a recessive X-linked form of muscular dystrophy. Examination of the relative targeting rate revealed that Crispr/Cas9 targeting could lead to mosaic mutations in up to 87% of the dystrophin alleles in monkey muscle. Moreover, CRISPR/Cas9 induced mutations in both male and female monkeys, with the markedly depleted dystrophin and muscle degeneration seen in early DMD. Our findings indicate that CRISPR/Cas9 can efficiently generate monkey models of human diseases, regardless of inheritance patterns. The presence of degenerated muscle cells in newborn Cas9-targeted monkeys suggests that therapeutic interventions at the early disease stage may be effective at alleviating the myopathy.

Figure 1.

DMD gene mutations generated by Cas9. (A) The location of targeted regions (exon 4 and exon 46) in the human DMD gene. Physical map of the dystrophin gene. Green boxes indicate exons, blue boxes indicate exons specific for each promoter (B1, first brain exon; M1, first muscle exon; P1, first Purkinje exon) and red boxes indicate promoters (BP, brain promoter; G1, general type promoter; MP, muscle promoter; PP, Purkinje promoter; S1, Schwann cell promoter). Not drawn to scale (Adapted from Cohen, N. and Muntoni, F. (2004) Multiple pathogenetic mechanisms in X linked dilated cardiomyopathy. Heart (British Cardiac Society), 90, 835–841). (B) Targeted sequences of the monkey DMD gene by Cas9 vectors.

Figure 2.

Monkey embryos injected with Cas9 mRNA and collected for PCR analysis. (A) The normal development of embryos after injection of Cas9 mRNA. (B) Cas9-mediated mutations in exon 4 and exon 46 of the monkey DMD gene. (C) Cas9-mediated DNA mutations in monkey embryos.

Figure 3.

Generation of live monkeys carrying mutations in the DMD gene. (A) CRISPR/Cas9-mediated targeting in monkeys. (B) Identified sequence mutations in full-term stillborn monkeys. (C) A photo of two monkeys at the age of 76 days, which were found to carry Cas9-mediated mutations. (D) Representative PCR analysis of mutations in exon 4 DMD gene of the DMD gene in full-term stillborn monkeys Mut-1 and Mut-2 and live monkeys (Mut-3, -4, -5 and -6). T7EN1 and enzymatic digestion were performed. WT, wild type; PC, positive control. (E) Identified sequence mutations in live monkeys.

Figure 4.

The relative rates of mosaic mutations in stillborn monkey tissues. (A) PCR products of the mutant alleles and the total DMD gene under the same PCR conditions. WT monkey tissues served as controls to show the specific amplification of mutant alleles of the DMD gene in monkey Mut-2. (B and C) Specific primers for each mutation (+20, +2 and −2) were able to amplify the mutant DNA from monkey Mut-1 (B) and Mut-2 (C), but not the WT monkey. Under the same PCR conditions, primers that could amplify both mutant and WT alleles were used to obtain total DMD gene products. (D and E) The levels (%) of each mutant alleles relative to total DMD alleles in different tissues were obtained by densitometry analysis of DNA bands in (B) and (C), respectively. Total mutations represent the sum of different mutations (+20, +2 and −2). The data are mean ± SE (n = 3 independent PCRs).

Figure 5.

Depletion of dystrophin by Cas9 in monkey tissues. (A) Immunohistochemical staining of muscle sections showing the reduced level of dystrophin in Mut-1 monkey muscle and the depletion of dystrophin in Mut-2 monkey muscle compared with wild-type stillborn monkey muscle. (B) High-magnification graphs of immunostained muscle sections from wild-type, Mut-1 and Mut-2 monkeys. (C) Western blot confirming the reduction of dystrophin in Mut-1 monkey muscle and depletion of dystrophin in Mut-2 monkey muscle. Scale bars: (A): 50 μm; (B): 20 μm.

Figure 6.

Hypertrophic myopathy caused by Cas9-mediated targeting of the DMD gene. Hematoxylin and eosin (H&E) staining of monkey muscle sections. (A) Low-magnification (×10) micrographs showing the clusters of hypertrophic muscle cells with an increase in the extent of the interstitial space in Mut-1 and Mut-2 monkeys. (B) Higher magnification micrograph of the DMD monkey muscle fibers shows multicentrally nuclei in hypertrophic muscle cells (arrows) compared with WT muscle. (C) Cross-sectional areas of normal muscle cells and hypertrophic muscle fibers containing centralized nuclei. Muscle sections from the stillborn wild-type monkey served as a control. Data are presented as mean ± SE from counting >300 cells in each group. *P < 0.05; **P < 0.01. Scale bars: 50 μm (left panel, ×10); 20 μm (right panel, ×40).