Yandi Gao

CRISPR-mediated Genome Editing Restores Dystrophin Expression and Function in mdx Mice

Duchenne muscular dystrophy (DMD) is a degenerative muscle disease caused by genetic mutations that lead to the disruption of dystrophin in muscle fibers. There is no curative treatment for this devastating disease. Clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) has emerged as a powerful tool for genetic manipulation and potential therapy. Here we demonstrate that CRIPSR-mediated genome editing efficiently excised a 23-kb genomic region on the X-chromosome covering the mutant exon 23 in a mouse model of DMD, and restored dystrophin expression and the dystrophin-glycoprotein complex at the sarcolemma of skeletal muscles in live mdx mice. Electroporation-mediated transfection of the Cas9/gRNA constructs in the skeletal muscles of mdx mice normalized the calcium sparks in response to osmotic shock. Adenovirus-mediated transduction of Cas9/gRNA greatly reduced the Evans blue dye uptake of skeletal muscles at rest and after downhill treadmill running. This study provides proof evidence for permanent gene correction in DMD.

In Vivo Genome Editing Restores Dystrophin Expression and Cardiac Function in Dystrophic Mice

Rationale: Duchenne muscular dystrophy is a severe inherited form of muscular dystrophy caused by mutations in the reading frame of the dystrophin gene disrupting its protein expression. Dystrophic cardiomyopathy is a leading cause of death in Duchenne muscular dystrophy patients, and currently no effective treatment exists to halt its progression. Recent advancement in genome editing technologies offers a promising therapeutic approach in restoring dystrophin protein expression. However, the impact of this approach on Duchenne muscular dystrophy cardiac function has yet to be evaluated. Therefore, we assessed the therapeutic efficacy of CRISPR (clustered regularly interspaced short palindromic repeats)-mediated genome editing on dystrophin expression and cardiac function in mdx/Utr+/− mice after a single systemic delivery of recombinant adeno-associated virus. Objective: To examine the efficiency and physiological impact of CRISPR-mediated genome editing on cardiac dystrophin expression and function in dystrophic mice. Methods and Results: Here, we packaged SaCas9 (clustered regularly interspaced short palindromic repeat–associated 9 from Staphylococcus aureus) and guide RNA constructs into an adeno-associated virus vector and systemically delivered them to mdx/Utr+/− neonates. We showed that CRIPSR-mediated genome editing efficiently excised the mutant exon 23 in dystrophic mice, and immunofluorescence data supported the restoration of dystrophin protein expression in dystrophic cardiac muscles to a level approaching 40%. Moreover, there was a noted restoration in the architecture of cardiac muscle fibers and a reduction in the extent of fibrosis in dystrophin-deficient hearts. The contractility of cardiac papillary muscles was also restored in CRISPR-edited cardiac muscles compared with untreated controls. Furthermore, our targeted deep sequencing results confirmed that our adeno-associated virus-CRISPR/Cas9 strategy was very efficient in deleting the ≈23 kb of intervening genomic sequences. Conclusions: This study provides evidence for using CRISPR-based genome editing as a potential therapeutic approach for restoring dystrophic cardiomyopathy structurally and functionally.

Empower multiplex cell and tissue-specific CRISPR-mediated gene manipulation with self-cleaving ribozymes and tRNA

Abstract Clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) system has emerged in recent years as a highly efficient RNA-guided gene manipulation platform. Simultaneous editing or transcriptional activation/suppression of different genes becomes feasible with the co-delivery of multiple guide RNAs (gRNAs). Here, we report that multiple gRNAs linked with self-cleaving ribozymes and/or tRNA could be simultaneously expressed from a single U6 promoter to exert genome editing of dystrophin and myosin binding protein C3 in human and mouse cells. Moreover, this strategy allows the expression of multiple gRNAs for synergistic transcription activation of follistatin when used with catalytically inactive dCas9-VP64 or dCas9-p300core fusions. Finally, the gRNAs linked by the self-cleaving ribozymes and tRNA could be expressed from RNA polymerase type II (pol II) promoters such as generic CMV and muscle/heart-specific MHCK7. This is particularly useful for in vivo applications when the packaging capacity of recombinant adeno-associated virus is limited while tissue-specific delivery of gRNAs and Cas9 is desired. Taken together, this study provides a novel strategy to enable tissue-specific expression of more than one gRNAs for multiplex gene editing from a single pol II promoter.

A novel rabbit model of Duchenne muscular dystrophy generated by CRISPR/Cas9

ABSTRACT Duchenne muscular dystrophy (DMD) is an X-linked muscle-wasting disorder caused by mutations in the dystrophin gene, with an incidence of 1 in 3500 in new male births. Mdx mice are widely used as an animal model for DMD. However, these mice do not faithfully recapitulate DMD patients in many aspects, rendering the preclinical findings in this model questionable. Although larger animal models of DMD, such as dogs and pigs, have been generated, usage of these animals is expensive and only limited to several facilities in the world. Here, we report the generation of a rabbit model of DMD by co-injection of Cas9 mRNA and sgRNA targeting exon 51 into rabbit zygotes. The DMD knockout (KO) rabbits exhibit the typical phenotypes of DMD, including severely impaired physical activity, elevated serum creatine kinase levels, and progressive muscle necrosis and fibrosis. Moreover, clear pathology was also observed in the diaphragm and heart at 5 months of age, similar to DMD patients. Echocardiography recording showed that the DMD KO rabbits had chamber dilation with decreased ejection fraction and fraction shortening. In conclusion, this novel rabbit DMD model generated with the CRISPR/Cas9 system mimics the histopathological and functional defects in DMD patients, and could be valuable for preclinical studies. This article has an associated First Person interview with the first author of the paper. Summary: The DMD KO rabbit engineered by CRISPR genome editing faithfully recapitulates the DMD pathologies, and could be a valuable tool for basic and translational studies to combat this disease.

A novel ANO5 splicing variant in a LGMD2L patient leads to production of a truncated aggregation-prone Ano5 peptide: A novel ANO5 splicing variant in LGMD2L

Mutations in ANO5 cause several human diseases including gnathodiaphyseal dysplasia 1 (GDD1), limb‐girdle muscular dystrophy 2L (LGMD2L), and Miyoshi myopathy 3 (MMD3). Previous work showed that complete genetic disruption of Ano5 in mice did not recapitulate human muscular dystrophy, while residual expression of mutant Ano5 in a gene trapped mouse developed muscular dystrophy with defective membrane repair. This suggests that truncated Ano5 expression may be pathogenic. Here, we screened a panel of commercial anti‐Ano5 antibodies using a recombinant adenovirus expressing human Ano5 with FLAG and YFP at the N‐ and C‐terminus, respectively. The monoclonal antibody (mAb) N421A/85 was found to specifically detect human Ano5 by immunoblotting and immunofluorescence staining. The antigen epitope was mapped to a region of 28 residues within the N‐terminus. Immunofluorescence staining of muscle cryosections from healthy control subjects showed that Ano5 is localized at the sarcoplasmic reticulum. The muscle biopsy from a LGMD2L patient homozygous for the c.191dupA mutation showed no Ano5 signal, confirming the specificity of the N421A/85 antibody. Surprisingly, strong Ano5 signal was detected in a patient with compound heterozygous mutations (c.191dupA and a novel splice donor site variant c.363 + 4A > G at the exon 6–intron 6 junction). Interestingly, insertion of the mutant intron 6, but not the wild‐type intron 6, into human ANO5 cDNA resulted in a major transcript that carried the first 158‐bp of intron 6. Transfection of the construct encoding the first 121 amino acids into C2C12 cells resulted in protein aggregate formation, suggesting that aggregate‐forming Ano5 peptide may contribute to the pathogenesis of muscular dystrophy.

Development of muscular dystrophy in a CRISPR-engineered mutant rabbit model with frame-disrupting ANO5 mutations

Limb girdle muscular dystrophy type 2L (LGMD2L) and Miyoshi myopathy type 3 (MMD3) are autosomal recessive muscular dystrophy caused by mutations in the gene encoding anoctamin-5 (ANO5), which belongs to the anoctamin protein family. Two independent lines of mice with complete disruption of ANO5 transcripts did not exhibit overt muscular dystrophy phenotypes; instead, one of these mice was observed to present with some abnormality in sperm motility. In contrast, a third line of ANO5-knockout (KO) mice with residual expression of truncated ANO5 expression was reported to display defective membrane repair and very mild muscle pathology. Many of the ANO5-related patients carry point mutations or small insertions/deletions (indels) in the ANO5 gene. To more closely mimic the human ANO5 mutations, we engineered mutant ANO5 rabbits via co-injection of Cas9 mRNA and sgRNA into the zygotes. CRISPR-mediated small indels in the exon 12 and/or 13 in the mutant rabbits lead to the development of typical signs of muscular dystrophy with increased serum creatine kinase (CK), muscle necrosis, regeneration, fatty replacement and fibrosis. This novel ANO5 mutant rabbit model would be useful in studying the disease pathogenesis and therapeutic treatments for ANO5-deficient muscular dystrophy.

Genome Editing Therapy for Duchenne Muscular Dystrophy

Abstract Duchenne muscular dystrophy (DMD) is a degenerative muscle disease caused by mutations in the DMD gene, which encodes the dystrophin protein. Recent advances in genome editing open doors for the development of potential “permanent” therapeutics. Today’s hottest genome editing tool, the CRISPR/Cas9 system, has recently been used to restore dystrophin reading frame and thus, its expression by deleting one or more exons encompassing the mutations in patient cells and in a mouse model of DMD. These approaches to restore dystrophin expression are highly promising, but many hurdles remain. This chapter summarizes the current state of genome editing therapeutic strategies for DMD and considerations for future development.

Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and living organisms from a wide variety of species. The CRISPR technology has also been explored as novel therapeutics for a number of human diseases. Proof-of-concept data are highly encouraging as exemplified by recent studies that demonstrate the feasibility and efficacy of gene editing-based therapeutic approach for Duchenne muscular dystrophy (DMD) using a murine model. In particular, intravenous and intraperitoneal injection of the recombinant adeno-associated virus (rAAV) serotype rh.74 (rAAVrh.74) has enabled efficient cardiac delivery of the Staphylococcus aureus CRISPR-associated protein 9 (SaCas9) and two guide RNAs (gRNA) to delete a genomic region with a mutant codon in exon 23 of mouse Dmd gene. This same approach can also be used to knock out the gene-of-interest and study their cardiac function in postnatal mice when the gRNA is designed to target the coding region of the gene. In this protocol, we show in detail how to engineer rAAVrh.74-CRISPR vector and how to achieve highly efficient cardiac delivery in neonatal mice.

20. A Novel Approach in the Treatment of Dystrophic Cardiomyopathy

Duchenne muscular dystrophy (DMD) is the most common muscular dystrophy with a worldwide incidence of one in 3500 live male births. It is caused by the lack of dystrophin, a critical muscle protein that connects the cytoskeleton and the extracellular matrix (ECM). Cardiomyopathy develops in at least 90% of patients and alone can shorten the life expectancy of DMD patients by at least 2 years and up to 40% of DMD patients eventually die from heart failure. Recently, RNA-guided, nuclease-mediated genome editing based on type II CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) system, has been emerged to alter the genome. In this study, we hypothesize that CRISPR-mediated genome editing could offer a novel therapy for DMD-associated cardiomyopathy in live mice. Two gRNA target sites were chosen from intron 20 and 23 of mouse Dmd. Co- transfection of the two gRNA with cas9 plasmids into mouse C2C12 cells resulted in the detection of a small PCR product as predicted, indicating successful CRISPR-mediated genome editing. DNA sequencing confirmed that the transcripts from C2C12 cells treated with gRNA/cas9 were formed due to successful deletion of exons 21-23 of mouse Dmd. Moreover, we injected the adenoviral vectors carrying GFP-2A-cas9 and gRNAs systemically and locally into the newborn pups. Four weeks after adenovirus transduction, dystrophin expression was restored in the heart muscles positive for GFP. Our PCR and western blotting data demonstrated that in-frame deletion of the genomic DNA covering exon 23 restored functional dystrophin expression in the hearts of mdx mice. Immunofluorescence staining also demonstrated that β-dystroglycan, which is normally located to the sarcolemma in healthy heart muscles via interaction with dystrophin-glycoprotein complex, was also restored at the sarcolemma of GFP-positive heart muscle fibers. These data provide the proof evidence of systemic restoration of dystrophin in the hearts of live mice.

577. Empower Multiplex CRISPR-Mediated Gene Manipulation with Self-Cleaving Ribozymes and tRNA

Clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) has been recently introduced as an efficient tool to edit the genome. Moreover, it can be targeted to specific gene loci by using single guide RNA (sgRNAs) for genetic manipulation and potential therapy. However, its targeting capability is often restricted by (gRNA) and generation of large deletions in genome by CRISPR requires expression of two distinct guide RNAs simultaneously. Here we report an innovative modification to increase the CRISPR/cas9 editing efficiency in the form of Ribozyme-flanked guide RNA/CRISPR system, which expresses two gRNA in equal molar amount. Our results show that this approach can be used to generate large DNA deletion with transfection of only one plasmid in human and mouse cells. Furthermore, we also used this system to target multiple transcriptional activators to a single promoter and the results showed that RNA level of targeting gene was statistically significantly up-regulated via Ribozyme gRNA/hCas9. Taken together our data, we prove that this strategy can be used in gene editing study broadly to enhance the targeting and the editing efficiency of CRISPR system.