Highly active zinc-finger nucleases by extended modular assembly.

Zinc-finger nucleases (ZFNs) are important tools for genome engineering. Despite intense interest by many academic groups, the lack of robust noncommercial methods has hindered their widespread use. The modular assembly (MA) of ZFNs from publicly available one-finger archives provides a rapid method to create proteins that can recognize a very broad spectrum of DNA sequences. However, three- and four-finger arrays often fail to produce active nucleases. Efforts to improve the specificity of the one-finger archives have not increased the success rate above 25%, suggesting that the MA method might be inherently inefficient due to its insensitivity to context-dependent effects. Here we present the first systematic study on the effect of array length on ZFN activity. ZFNs composed of six-finger MA arrays produced mutations at 15 of 21 (71%) targeted loci in human and mouse cells. A novel drop-out linker scheme was used to rapidly assess three- to six-finger combinations, demonstrating that shorter arrays could improve activity in some cases. Analysis of 268 array variants revealed that half of MA ZFNs of any array composition that exceed an ab initio B-score cutoff of 15 were active. These results suggest that, when used appropriately, MA ZFNs are able to target more DNA sequences with higher success rates than other current methods.

Synergistic and tunable human gene activation by combinations of synthetic transcription factors.

Mammalian genes are regulated by the cooperative and synergistic actions of many transcription factors. In this study we recapitulate this complex regulation in human cells by targeting endogenous gene promoters, including regions of closed chromatin upstream of silenced genes, with combinations of engineered transcription activator-like effectors (TALEs). These combinations of TALE transcription factors induced substantial gene activation and allowed tuning of gene expression levels that will broadly enable synthetic biology, gene therapy and biotechnology.

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.

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.

Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector.

Engineered DNA-binding proteins that manipulate the human genome and transcriptome have enabled rapid advances in biomedical research. In particular, the RNA-guided CRISPR/Cas9 system has recently been engineered to create site-specific double-strand breaks for genome editing or to direct targeted transcriptional regulation. A unique capability of the CRISPR/Cas9 system is multiplex genome engineering by delivering a single Cas9 enzyme and two or more single guide RNAs (sgRNAs) targeted to distinct genomic sites. This approach can be used to simultaneously create multiple DNA breaks or to target multiple transcriptional activators to a single promoter for synergistic enhancement of gene induction. To address the need for uniform and sustained delivery of multiplex CRISPR/Cas9-based genome engineering tools, we developed a single lentiviral system to express a Cas9 variant, a reporter gene and up to four sgRNAs from independent RNA polymerase III promoters that are incorporated into the vector by a convenient Golden Gate cloning method. Each sgRNA is efficiently expressed and can mediate multiplex gene editing and sustained transcriptional activation in immortalized and primary human cells. This delivery system will be significant to enabling the potential of CRISPR/Cas9-based multiplex genome engineering in diverse cell types.

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.

Gene targeting to the ROSA26 locus directed by engineered zinc finger nucleases.

Targeted gene addition to mammalian genomes is central to biotechnology, basic research and gene therapy. For example, gene targeting to the ROSA26 locus by homologous recombination in embryonic stem cells is commonly used for mouse transgenesis to achieve ubiquitous and persistent transgene expression. However, conventional methods are not readily adaptable to gene targeting in other cell types. The emerging zinc finger nuclease (ZFN) technology facilitates gene targeting in diverse species and cell types, but an optimal strategy for engineering highly active ZFNs is still unclear. We used a modular assembly approach to build ZFNs that target the ROSA26 locus. ZFN activity was dependent on the number of modules in each zinc finger array. The ZFNs were active in a variety of cell types in a time- and dose-dependent manner. The ZFNs directed gene addition to the ROSA26 locus, which enhanced the level of sustained gene expression, the uniformity of gene expression within clonal cell populations and the reproducibility of gene expression between clones. These ZFNs are a promising resource for cell engineering, mouse transgenesis and pre-clinical gene therapy studies. Furthermore, this characterization of the modular assembly method provides general insights into the implementation of the ZFN technology.

Feline lectin activity is critical for the cellular entry of feline infectious peritonitis virus.

Feline infectious peritonitis is a lethal disease of felids caused by systemic infection with a feline coronavirus. Here, we report identification and analysis of the feline homologue to the human lectin DC-SIGN and show that it is a coreceptor for virulent strains of serotype 1 and serotype 2 feline coronaviruses.

In Vivo Targeting of Clostridioides difficile Using Phage-Delivered CRISPR-Cas3 Antimicrobials.

Clostridioides difficile is an important nosocomial pathogen that causes approximately 500,000 cases of C. difficile infection (CDI) and 29,000 deaths annually in the United States. Antibiotic use is a major risk factor for CDI because broad-spectrum antimicrobials disrupt the indigenous gut microbiota, decreasing colonization resistance against C. difficile Vancomycin is the standard of care for the treatment of CDI, likely contributing to the high recurrence rates due to the continued disruption of the gut microbiota. Thus, there is an urgent need for the development of novel therapeutics that can prevent and treat CDI and precisely target the pathogen without disrupting the gut microbiota. Here, we show that the endogenous type I-B CRISPR-Cas system in C. difficile can be repurposed as an antimicrobial agent by the expression of a self-targeting CRISPR that redirects endogenous CRISPR-Cas3 activity against the bacterial chromosome. We demonstrate that a recombinant bacteriophage expressing bacterial genome-targeting CRISPR RNAs is significantly more effective than its wild-type parent bacteriophage at killing C. difficile both in vitro and in a mouse model of CDI. We also report that conversion of the phage from temperate to obligately lytic is feasible and contributes to the therapeutic suitability of intrinsic C. difficile phages, despite the specific challenges encountered in the disease phenotypes of phage-treated animals. Our findings suggest that phage-delivered programmable CRISPR therapeutics have the potential to leverage the specificity and apparent safety of phage therapies and improve their potency and reliability for eradicating specific bacterial species within complex communities, offering a novel mechanism to treat pathogenic and/or multidrug-resistant organisms.IMPORTANCEClostridioides difficile is a bacterial pathogen responsible for significant morbidity and mortality across the globe. Current therapies based on broad-spectrum antibiotics have some clinical success, but approximately 30% of patients have relapses, presumably due to the continued perturbation to the gut microbiota. Here, we show that phages can be engineered with type I CRISPR-Cas systems and modified to reduce lysogeny and to enable the specific and efficient targeting and killing of C. difficilein vitro and in vivo. Additional genetic engineering to disrupt phage modulation of toxin expression by lysogeny or other mechanisms would be required to advance a CRISPR-enhanced phage antimicrobial for C. difficile toward clinical application. These findings provide evidence into how phage can be combined with CRISPR-based targeting to develop novel therapies and modulate microbiomes associated with health and disease.

The Development of TALE Nucleases for Biotechnology.

The development of a facile genome engineering technology based on transcription activator-like effector nucleases (TALENs) has led to significant advances in diverse areas of science and medicine. In this review, we provide a broad overview of the development of TALENs and the use of this technology in basic science, biotechnology, and biomedical applications. This includes the discovery of DNA recognition by TALEs, engineering new TALE proteins to diverse targets, general advances in nuclease-based editing strategies, and challenges that are specific to various applications of the TALEN technology. We review examples of applying TALENs for studying gene function and regulation, generating disease models, and developing gene therapies. The current status of genome editing and future directions for other uses of these technologies are also discussed.

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

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.

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.

Advances in targeted genome editing.

New technologies have recently emerged that enable targeted editing of genomes in diverse systems. This includes precise manipulation of gene sequences in their natural chromosomal context and addition of transgenes to specific genomic loci. This progress has been facilitated by advances in engineering targeted nucleases with programmable, site-specific DNA-binding domains, including zinc finger proteins and transcription activator-like effectors (TALEs). Recent improvements have enhanced nuclease performance, accelerated nuclease assembly, and lowered the cost of genome editing. These advances are driving new approaches to many areas of biotechnology, including biopharmaceutical production, agriculture, creation of transgenic organisms and cell lines, and studies of genome structure, regulation, and function. Genome editing is also being investigated in preclinical and clinical gene therapies for many diseases.