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CRISPR applications for Duchenne muscular dystrophy: From animal models to potential therapies.
CRISPR gene-editing technology creates precise and permanent modifications to DNA. It has significantly advanced our ability to generate animal disease models for use in biomedical research and also has potential to revolutionize the treatment of genetic disorders. Duchenne muscular dystrophy (DMD) is a monogenic muscle-wasting disease that could potentially benefit from the development of CRISPR therapy. It is commonly associated with mutations that disrupt the reading frame of the DMD gene that encodes dystrophin, an essential scaffolding protein that stabilizes striated muscles and protects them from contractile-induced damage. CRISPR enables the rapid generation of various animal models harboring mutations that closely simulates the wide variety of mutations observed in DMD patients. These models provide a platform for the testing of sequence-specific interventions like CRISPR therapy that aim to reframe or skip DMD mutations to restore functional dystrophin expression. This article is categorized under: Congenital Diseases > Genetics/Genomics/Epigenetics.
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Structures of Cas9 endonucleases reveal RNA-mediated conformational activation.
Type II CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems use an RNA-guided DNA endonuclease, Cas9, to generate double-strand breaks in invasive DNA during an adaptive bacterial immune response. Cas9 has been harnessed as a powerful tool for genome editing and gene regulation in many eukaryotic organisms. We report 2.6 and 2.2 angstrom resolution crystal structures of two major Cas9 enzyme subtypes, revealing the structural core shared by all Cas9 family members. The architectures of Cas9 enzymes define nucleic acid binding clefts, and single-particle electron microscopy reconstructions show that the two structural lobes harboring these clefts undergo guide RNA-induced reorientation to form a central channel where DNA substrates are bound. The observation that extensive structural rearrangements occur before target DNA duplex binding implicates guide RNA loading as a key step in Cas9 activation.
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Optimization and validation of CAR transduction into human primary NK cells using CRISPR and AAV.
Human primary natural killer (NK) cells are being widely advanced for cancer immunotherapy. However, methods for gene editing of these cells have suffered low transduction rates, high cell death, and loss of transgene expression after expansion. Here, we developed a highly efficient method for site-specific gene insertion in NK cells using CRISPR (Cas9/RNP) and AAVs. We compared AAV vectors designed to mediate gene insertion by different DNA repair mechanisms, homology arm lengths, and virus concentrations. We then validated the method for site-directed gene insertion of CD33-specific CARs into primary human NK cells. CAR transduction was efficient, its expression remained stable after expansion, and it improved efficacy against AML targets.
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Discovery, classification and application of the CPISPR-Cas13 system.
BACKGROUND: The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system is an acquired immune system of bacteria and archaea. Continued research has resulted in the identification of other Cas13 proteins. OBJECTIVE: This review briefly describes the discovery, classification, and application of the CRISPR-Cas13 system, including recent technological advances in addition to factors affecting system performance. METHODS: Cas13-based molecular therapy of human, animal, and plant transcriptomes was discussed, including regulation of gene expression to combat pathogenic RNA viruses. In addition, the latest progress, potential shortcomings, and challenges of the CRISPR-Cas system for treatment of animal and plant diseases are reviewed. RESULTS: The CRISPR-Cas system VI is characterized by two RNA-guided higher eukaryotes and prokaryotes nucleotide-binding domains. CRISPR RNA can cleave specific RNA through the interaction between the stem-loop rich chain of uracil residues and the Cas13a protein. The CRISPR-Cas13 system has been applied for gene editing in animal and plant cells, in addition to biological detection via accurate targeting of single-stranded RNA. CONCLUSION: The CRISPR-Cas13 system offers a high-throughput and convenient technology for detection of viruses and potentially the development of anti-cancer drugs in the near future.
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Silencing of the Mutant Huntingtin Gene through CRISPR-Cas9 Improves the Mitochondrial Biomarkers in an In Vitro Model of Huntington's Disease.
During the 25-year history of the American Society for Neural Therapy and Repair (ASNTR) there have been several breakthroughs in the area of neurotherapeutics, which was the case during the 2014-2105 year when one of us (GLD) had the privilege of serving as its president. During that year, the use of a newly developed gene-editing tool, the CRISPR-Cas9 system, started to skyrocket. Although scientists unraveled the use of "clustered regularly interspaced short palindromic repeats" (CRISPR) and its associated genes from the Cas family as an evolved mechanism of some bacterial and archaeal genomes to protect themselves from being hijacked by invasive viral genes, its use as a therapeutic tool was not fully appreciated until further research revealed how this system operated and how it might be developed technologically to manipulate genes of any species. By 2015, this technology had exploded to the point that close to 2,000 papers that used this technology were published during that year alone.
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Phenylboronic acid-functionalized polyaminoglycoside as an effective CRISPR/Cas9 delivery system.
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing technology is a promising approach for cancer therapy, and its application practice urgently requires a safe and effective gene carrier. In this work, we focus on the design of a phenylboronic acid (PBA)-functionalized, disulfide bonded branched polyaminoglycoside (SS-HPT-P) as a robust delivery vector of the CRISPR-Cas9 system. SS-HPT-P showed great tumor-targeting performance, reduction-responsive degradability, and gene transfection ability. The typical pCas9-surv (one CRISPR-Cas9 plasmid that targets and knocks out the survivin gene) delivery mediated by SS-HPT-P exhibited gene editing performance in the A549 cell line, confirming the feasibility of SS-HPT-P to effectively deliver the CRISPR-Cas9 system. SS-HPT-P/pCas9-surv could effectively inhibit the proliferation of tumor cells both in vitro and in vivo, suggesting the potential of PBA-functionalized nanocarriers for cancer gene therapy. The present work provides a promising approach for the treatment of malignant tumors.
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Evaluation and Reduction of CRISPR Off-Target Cleavage Events.
Introduction of CRISPR/Cas9 methods (clustered regularly interspaced short palindromic repeats, CRISPR-associated protein 9) have led to a huge surge in the use of precision genome editing for research applications. Translational medical efforts are likewise rapidly progressing, and Phase I clinical trials using these techniques have already started. As with any new technology that is applied to medical therapeutics, risks must be carefully defined and steps taken to mitigate side effects wherever possible. Effective methods are now available that permit identification of off-target cleavage events, a major class of potential side effects seen in mammalian genome editing. Off-target prediction algorithms are improving and have utility, but are insufficient to use alone. Empiric methods to define the off-target profile must also be used. Once defined, the frequency of off-target cleavage can be minimized using methods that limit the duration of exposure of the genome to the active genome editing complex, for example, using the ribonucleoprotein (RNP) approach. In addition, Cas9 mutants have been developed that markedly reduce the rate of off-target cleavage compared to the wild-type enzyme. Use of these new tools should become standard practice for medical applications.
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Advantages of using the CRISPR/Cas9 system of genome editing to investigate male reproductive mechanisms using mouse models.
Gene disruption technology has long been beneficial for the study of male reproductive biology. However, because of the time and cost involved, this technology was not a viable method except in specialist laboratories. The advent of the CRISPR/Cas9 system of gene disruption has ushered in a new era of genetic investigation. Now, it is possible to generate gene-disrupted mouse models in very little time and at very little cost. This Highlight article discusses the application of this technology to study the genetics of male fertility and looks at some of the future uses of this system that could be used to reveal the essential and nonessential genetic components of male reproductive mechanisms.
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CRISPR-mediated gene modification of hematopoietic stem cells with beta-thalassemia IVS-1-110 mutation.
BACKGROUND: β-Thalassemias represent a group of genetic disorders caused by human hemoglobin beta (HBB) gene mutations. The radical curative approach is to correct the mutations causing the disease. CRISPR-CAS9 is a novel gene-editing technology that can be used auspiciously for the treatment of these disorders. The study aimed to investigate the utility of CRISPR-CAS9 for gene modification of hematopoietic stem cells in β-thalassemia with IVS-1-110 mutation. METHODS AND RESULTS: We successfully isolated CD34+ cells from peripheral blood of β-thalassemia patients with IVS-1-110 mutation. The cells were transfected with Cas9 endonuclease together with guide RNA to create double-strand breaks and knock out the mutation. The mutation-corrected CD34+ cells were subjected to erythroid differentiation by culturing in complete media containing erythropoietin. CONCLUSION: CRISPR/Cas-9 is an effective tool for gene therapy that will broaden the spectrum of therapy and potentially improve the outcomes of β-thalassemia.
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CRISPR-Cas based targeting of host and viral genes as an antiviral strategy.
Viral infections in human are leading cause of mortality and morbidity across the globe. Several viruses (including HIV and Herpesvirus), have evolved ingenious strategies to evade host-immune system and persist life-long. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) is an ancient antiviral system recently discovered in bacteria that has shown tremendous potential as a precise, invariant genome editing tool. Using CRISPR-Cas based system to activate host defenses or genetic modification of viral genome can provide novel, exciting and successful antiviral mechanisms and treatment modalities. In this review, we will provide progress on the CRISPR-Cas based antiviral approaches that facilitate clearance of virus-infected cells and/or prohibit virus infection or replication. We will discuss on the possibilities of CRIPSR-Cas as prophylaxis and therapy in viral infections and review the challenges of this potent gene editing technology.
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