Pratiksha I. Thakore

Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers

Technologies that enable targeted manipulation of epigenetic marks could be used to precisely control cell phenotype or interrogate the relationship between the epigenome and transcriptional control. Here we describe a programmable, CRISPR-Cas9-based acetyltransferase consisting of the nuclease-null dCas9 protein fused to the catalytic core of the human acetyltransferase p300. The fusion protein catalyzes acetylation of histone H3 lysine 27 at its target sites, leading to robust transcriptional activation of target genes from promoters and both proximal and distal enhancers. Gene activation by the targeted acetyltransferase was highly specific across the genome. In contrast to previous dCas9-based activators, the acetyltransferase activates genes from enhancer regions and with an individual guide RNA. We also show that the core p300 domain can be fused to other programmable DNA-binding proteins. These results support targeted acetylation as a causal mechanism of transactivation and provide a robust tool for manipulating gene regulation.

RNA-guided gene activation by CRISPR-Cas9–based transcription factors

Technologies for engineering synthetic transcription factors have enabled many advances in medical and scientific research. In contrast to existing methods based on engineering of DNA-binding proteins, we created a Cas9-based transactivator that is targeted to DNA sequences by guide RNA molecules. Coexpression of this transactivator and combinations of guide RNAs in human cells induced specific expression of endogenous target genes, demonstrating a simple and versatile approach for RNA-guided gene activation.

In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy.

Editing can help build stronger muscles Much of the controversy surrounding the gene-editing technology called CRISPR/Cas9 centers on the ethics of germline editing of human embryos to correct disease-causing mutations. For certain disorders such as muscular dystrophy, it may be possible to achieve therapeutic benefit by editing the faulty gene in somatic cells. In proof-of-concept studies, Long et al., Nelson et al., and Tabebordbar et al. used adeno-associated virus-9 to deliver the CRISPR/Cas9 gene-editing system to young mice with a mutation in the gene coding for dystrophin, a muscle protein deficient in patients with Duchenne muscular dystrophy. Gene editing partially restored dystrophin protein expression in skeletal and cardiac muscle and improved skeletal muscle function. Science, this issue p. 400, p. 403, p. 407 Gene editing via CRISPR-Cas9 restores dystrophin protein and improves muscle function in mouse models of muscular dystrophy. Duchenne muscular dystrophy (DMD) is a devastating disease affecting about 1 out of 5000 male births and caused by mutations in the dystrophin gene. Genome editing has the potential to restore expression of a modified dystrophin gene from the native locus to modulate disease progression. In this study, adeno-associated virus was used to deliver the clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 system to the mdx mouse model of DMD to remove the mutated exon 23 from the dystrophin gene. This includes local and systemic delivery to adult mice and systemic delivery to neonatal mice. Exon 23 deletion by CRISPR-Cas9 resulted in expression of the modified dystrophin gene, partial recovery of functional dystrophin protein in skeletal myofibers and cardiac muscle, improvement of muscle biochemistry, and significant enhancement of muscle force. This work establishes CRISPR-Cas9–based genome editing as a potential therapy to treat DMD.

Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory elements

Epigenome editing with the CRISPR (clustered, regularly interspaced, short palindromic repeats)-Cas9 platform is a promising technology for modulating gene expression to direct cell phenotype and to dissect the causal epigenetic mechanisms of gene regulation. Fusions of nuclease-inactive dCas9 to the Krüppel-associated box (KRAB) repressor (dCas9-KRAB) can silence target gene expression, but the genome-wide specificity and the extent of heterochromatin formation catalyzed by dCas9-KRAB are not known. We targeted dCas9-KRAB to the HS2 enhancer, a distal regulatory element that orchestrates the expression of multiple globin genes, and observed highly specific induction of H3K9 trimethylation (H3K9me3) at the enhancer and decreased chromatin accessibility of both the enhancer and its promoter targets. Targeted epigenetic modification of HS2 silenced the expression of multiple globin genes, with minimal off-target changes in global gene expression. These results demonstrate that repression mediated by dCas9-KRAB is sufficiently specific to disrupt the activity of individual enhancers via local modification of the epigenome.

Multiplex CRISPR/Cas9-based genome editing for correction of dystrophin mutations that cause Duchenne muscular dystrophy

The CRISPR/Cas9 genome editing platform is a promising technology to correct the genetic basis of hereditary diseases. The versatility, efficiency, and multiplexing capabilities of the CRISPR/Cas9 system enable a variety of otherwise challenging gene correction strategies. Here we use the CRISPR/Cas9 system to restore the expression of the dystrophin gene in cells carrying dystrophin mutations that cause Duchenne muscular dystrophy (DMD). We design single or multiplexed sgRNAs to restore the dystrophin reading frame by targeting the mutational hotspot at exons 45–55 and introducing shifts within exons or deleting one or more exons. Following gene editing in DMD patient myoblasts, dystrophin expression is restored in vitro. Human dystrophin is also detected in vivo after transplantation of genetically corrected patient cells into immunodeficient mice. Importantly, the unique multiplex gene editing capabilities of the CRISPR/Cas9 system facilitate the generation of a single large deletion that can correct up to 62% of DMD mutations.

Editing the epigenome: technologies for programmable transcription and epigenetic modulation

Gene regulation is a complex and tightly controlled process that defines cell identity, health and disease, and response to pharmacologic and environmental signals. Recently developed DNA-targeting platforms, including zinc finger proteins, transcription activator-like effectors (TALEs) and the clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 system, have enabled the recruitment of transcriptional modulators and epigenome-modifying factors to any genomic site, leading to new insights into the function of epigenetic marks in gene expression. Additionally, custom transcriptional and epigenetic regulation is facilitating refined control over cell function and decision making. The unique properties of the CRISPR-Cas9 system have created new opportunities for high-throughput genetic screens and multiplexing targets to manipulate complex gene expression patterns. This Review summarizes recent technological developments in this area and their application to biomedical challenges. We also discuss remaining limitations and necessary future directions for this field.

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

Genome editing with engineered nucleases has recently emerged as an approach to correct genetic mutations by enhancing homologous recombination with a DNA repair template. However, many genetic diseases, such as Duchenne muscular dystrophy (DMD), can be treated simply by correcting a disrupted reading frame. We show that genome editing with transcription activator-like effector nucleases (TALENs), without a repair template, can efficiently correct the reading frame and restore the expression of a functional dystrophin protein that is mutated in DMD. TALENs were engineered to mediate highly efficient gene editing at exon 51 of the dystrophin gene. This led to restoration of dystrophin protein expression in cells from Duchenne patients, including skeletal myoblasts and dermal fibroblasts that were reprogrammed to the myogenic lineage by MyoD. Finally, exome sequencing of cells with targeted modifications of the dystrophin locus showed no TALEN-mediated off-target changes to the protein-coding regions of the genome, as predicted by in silico target site analysis. This strategy integrates the rapid and robust assembly of active TALENs with an efficient gene-editing method for the correction of genetic diseases caused by mutations in non-essential coding regions that cause frameshifts or premature stop codons.

Correction of dystrophin expression in cells from Duchenne muscular dystrophy patients through genomic excision of exon 51 by zinc finger nucleases.

Duchenne muscular dystrophy (DMD) is caused by genetic mutations that result in the absence of dystrophin protein expression. Oligonucleotide-induced exon skipping can restore the dystrophin reading frame and protein production. However, this requires continuous drug administration and may not generate complete skipping of the targeted exon. In this study, we apply genome editing with zinc finger nucleases (ZFNs) to permanently remove essential splicing sequences in exon 51 of the dystrophin gene and thereby exclude exon 51 from the resulting dystrophin transcript. This approach can restore the dystrophin reading frame in ~13% of DMD patient mutations. Transfection of two ZFNs targeted to sites flanking the exon 51 splice acceptor into DMD patient myoblasts led to deletion of this genomic sequence. A clonal population was isolated with this deletion and following differentiation we confirmed loss of exon 51 from the dystrophin mRNA transcript and restoration of dystrophin protein expression. Furthermore, transplantation of corrected cells into immunodeficient mice resulted in human dystrophin expression localized to the sarcolemmal membrane. Finally, we quantified ZFN toxicity in human cells and mutagenesis at predicted off-target sites. This study demonstrates a powerful method to restore the dystrophin reading frame and protein expression by permanently deleting exons.

Enhanced MyoD-induced transdifferentiation to a myogenic lineage by fusion to a potent transactivation domain.

Genetic reprogramming holds great potential for disease modeling, drug screening, and regenerative medicine. Genetic reprogramming of mammalian cells is typically achieved by forced expression of natural transcription factors that control master gene networks and cell lineage specification. However, in many instances, the natural transcription factors do not induce a sufficiently robust response to completely reprogram cell phenotype. In this study, we demonstrate that protein engineering of the master transcription factor MyoD can enhance the conversion of human dermal fibroblasts and adult stem cells to a skeletal myocyte phenotype. Fusion of potent transcriptional activation domains to MyoD led to increased myogenic gene expression, myofiber formation, cell fusion, and global reprogramming of the myogenic gene network. This work supports a general strategy for synthetically enhancing the direct conversion between cell types that can be applied in both synthetic biology and regenerative medicine.

RNA-guided transcriptional silencing in vivo with S. aureus CRISPR-Cas9 repressors

CRISPR-Cas9 transcriptional repressors have emerged as robust tools for disrupting gene regulation in vitro but have not yet been adapted for systemic delivery in adult animal models. Here we describe a Staphylococcus aureus Cas9-based repressor (dSaCas9KRAB) compatible with adeno-associated viral (AAV) delivery. To evaluate dSaCas9KRAB efficacy for gene silencing in vivo, we silenced transcription of Pcsk9, a regulator of cholesterol levels, in the liver of adult mice. Systemic administration of a dual-vector AAV8 system expressing dSaCas9KRAB and a Pcsk9-targeting guide RNA (gRNA) results in significant reductions of serum Pcsk9 and cholesterol levels. Despite a moderate host response to dSaCas9KRAB expression, Pcsk9 repression is maintained for 24 weeks after a single treatment, demonstrating the potential for long-term gene silencing in post-mitotic tissues with dSaCas9KRAB. In vivo programmable gene silencing enables studies that link gene regulation to complex phenotypes and expands the CRISPR-Cas9 perturbation toolbox for basic research and gene therapy applications.Repression of gene transcription using CRISPR-Cas9 has been achieved in vitro but not for delivery into adult animal models. Here, the authors use AAV8 to deliver the transcriptional repressor dSaCas9KRAB to the cholesterol regulator Pcsk9, and show repression up to 24 weeks and reduced cholesterol levels in mice.