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Gene editing in T cell therapy.
The adoptive transfer of engineered T cells for the treatment of cancer, autoimmunity, and infectious disease is a rapidly growing field that has shown great promise. Gene editing holds tremendous potential for further improvements of T cell therapy. Here we review the applications of gene editing in various T cell therapies, focusing on antiviral strategies and cancer immunotherapies, and discuss the challenges and future prospects.
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Gene editing in hematopoietic stem cells by co-delivery of Cas9/sgRNA ribonucleoprotein and templates for homology-directed repair in 'all-in-one' lentivirus-derived nanoparticles.
Repair of double-strand DNA breaks generated by site-directed endonucleases, like Cas9, is the hallmark of gene editing based on homology-directed repair (HDR). HDR uses an exogenous DNA template to restore the cleaved DNA sequence and can facilitate specific gene corrections as well as insertion of genes or partial complementary DNA (cDNA) sequences. For CRISPR/Cas-directed gene editing, co-administration of the Cas9/single guide RNA (sgRNA) ribonucleoprotein (RNP) complex and a DNA template typically involves two different delivery strategies or different types of vehicles. This requires exquisite timing of delivery and may potentially challenge safety and therapeutic applicability. There is a need therefore for technologies that can ferry complete editing tool kits into cells. Here, we demonstrate the use of lentivirus-derived nanoparticles (LVNPs) to transport both RNP complexes and vector RNA, which upon reverse transcription serves as a repair template for HDR-directed gene editing. Such 'all-in-one' LVNPs support targeted gene insertion with reduced off-target effects relative to nucleofection procedures. We show potent editing in the HBB gene in human erythroid progenitor cells as well as HDR-directed editing in hematopoietic stem and progenitor cells. Our findings mark a first step toward using a single virus-derived vehicle for delivering a full HDR gene editing kit.
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Production of Duchenne muscular dystrophy cellular model using CRISPR-Cas9 exon deletion strategy.
Duchenne Muscular Dystrophy (DMD) is a progressive muscle wasting disorder caused by loss-of-function mutations in the dystrophin gene. Although the search for a definitive cure has failed to date, extensive efforts have been made to introduce effective therapeutic strategies. Gene editing technology is a great revolution in biology, having an immediate application in the generation of research models. DMD muscle cell lines are reliable sources to evaluate and optimize therapeutic strategies, in-depth study of DMD pathology, and screening the effective drugs. However, only a few immortalized muscle cell lines with DMD mutations are available. In addition, obtaining muscle cells from patients also requires an invasive muscle biopsy. Mostly DMD variants are rare, making it challenging to identify a patient with a particular mutation for a muscle biopsy. To overcome these challenges and generate myoblast cultures, we optimized a CRISPR/Cas9 gene editing approach to model the most common DMD mutations that include approximately 28.2% of patients. GAP-PCR and sequencing results show the ability of the CRISPR-Cas9 system to efficient deletion of mentioned exons. We showed producing truncated transcript due to the targeted deletion by RT-PCR and sequencing. Finally, mutation-induced disruption of dystrophin protein expression was confirmed by western blotting. All together, we successfully created four immortalized DMD muscle cell lines and showed the efficacy of the CRISPR-Cas9 system for the generation of immortalized DMD cell models with the targeted deletions.
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Visual Assay for Gene Editing Using a CRISPR/Cas9 System in Carrot Cells.
The development of the Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas9) system has advanced genome editing and has become widely adopted for this purpose in many species. Its efficient use requires the method adjustment and optimization. Here, we show the use of a model carrot callus system for demonstrating gene editing via CRISPR/Cas9 targeted mutagenesis. The system relies on the utilization of carrot tissue accumulating anthocyanin pigments responsible for a deep purple cell color and generation of knockout mutations in the flavanone-3-hydroxylase (F3H) gene in the anthocyanin biosynthesis pathway. F3H mutant cells targeted by Cas9/gRNA complexes are not able to synthesize anthocyanins and remain white, easily visually distinguished from purple wild-type cells. Mutations are either small indels or larger chromosomal deletions that can be identified by restriction fragment analysis and sequencing. This feasible system can also be applied for validating efficiency of CRISPR/Cas9 vectors.
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Manipulating plant RNA-silencing pathways to improve the gene editing efficiency of CRISPR/Cas9 systems.
BACKGROUND: The CRISPR/Cas9 system, composed of a single-guide RNA for target recognition and a Cas9 protein for DNA cleavage, has the potential to revolutionize agriculture as well as medicine. Even though extensive work has been done to improve the gene editing activity of CRISPR/Cas9, little is known about the regulation of this bacterial system in eukaryotic host cells, especially at the post-transcriptional level. RESULTS: Here, we evaluate the expression levels of the two CRISPR/Cas9 components and the gene editing efficiency in a set of Arabidopsis mutants involved in RNA silencing. We find that mutants defective in the post-transcriptional gene-silencing pathway display significantly higher Cas9 and sgRNA transcript levels, resulting in higher mutagenesis frequencies than wild-type controls. Accordingly, silencing of AGO1 by introduction of an AGO1-RNAi cassette into the CRISPR/Cas9 vector provides an increase in gene editing efficiency. Co-expression of the viral suppressor p19 from the tomato bushy stunt virus to suppress the plant RNA-silencing pathway shows a strong correlation between the severity of the phenotypic effects caused by p19 and the gene editing efficiency of the CRISPR/Cas9 system for two different target genes, AP1 and TT4. CONCLUSIONS: This system has useful practical applications in facilitating the detection of CRISPR/Cas9-induced mutations in T1 plants as well as the identification of transgene-free T2 plants by simple visual observation of the symptom severity caused by p19. Our study shows that CRISPR/Cas9 gene editing efficiency can be improved by reducing RNA silencing in plants.
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Focus on CRISPR tools and therapies.
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Outlook on the Security and Potential Improvements of CRISPR-Cas9.
Gene editing technology is regarded as a good news to save patients with genetic diseases because of its significant function of specifically changing genetic information. From zinc-finger proteins to transcription activator-like effector protein nucleases gene editing tools are constantly updated. At the same time, scientists are constantly developing a variety of new gene editing therapy strategies, in order to promote gene editing therapy from various aspects and realize the maturity of the technology as soon as possible. In 2016, CRISPR-Cas9-mediated CAR-T therapy was the first to enter the clinical trial stage, indicating that the use of CRISPR-Cas system as the blade of the genetic lancet to save patients is officially on the schedule. The first challenge to achieve this exciting goal is to improve the security of the technology. This review will introduce the gene security issues faced by the CRISPR system as a clinical treatment tool, the current safer delivery methods and the newly developed CRISPR editing tools with higher precision. Many reviews summarize the means of improving the security of gene editing therapy and the comprehensive delivery method, while few articles focus on the threat of gene editing to the genomic security of the treatment target. Therefore, this review focuses on the risks brought by gene editing therapy to the patient genome, which provides a broader perspective for exploring and improving the security of gene editing therapy from two aspects of delivery system and CRISPR editing tools.
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China's regulatory change toward genome-edited crops.
China's newly issued genome-edited crop safety assessment guidelines do not entirely deviate from the existing genetically modified regulatory regimes. However, there is an urgent need to reform the genome-edited regulatory framework based on scientific values and a novel regulatory philosophy that balances diverse values to enhance public trust.
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In vivo gene editing of CAMKIID: out with the bad and in with the good.
The ability to change an organism's DNA through gene editing is of great importance for the prevention and treatment of genetic and acquired diseases. Rapid progress has been made during the last decade due to the discovery and refinement of CRISPR/Cas9 as an accurate, fast, and reliable genome editing technique. In this issue of the JCI, Lebek et al. present the culmination from a line of work in the Olson laboratory focused on in vivo gene editing of CAMK2D. The paper presents a combined state-of-the-art gene therapy approach that demonstrates how gene therapy can yield cardioprotection in a mouse model and takes notable steps toward potential applicability in patients.
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A potential paradigm in CRISPR/Cas systems delivery: at the crossroad of microalgal gene editing and algal-mediated nanoparticles.
Microalgae as the photosynthetic organisms offer enormous promise in a variety of industries, such as the generation of high-value byproducts, biofuels, pharmaceuticals, environmental remediation, and others. With the rapid advancement of gene editing technology, CRISPR/Cas system has evolved into an effective tool that revolutionised the genetic engineering of microalgae due to its robustness, high target specificity, and programmability. However, due to the lack of robust delivery system, the efficacy of gene editing is significantly impaired, limiting its application in microalgae. Nanomaterials have become a potential delivery platform for CRISPR/Cas systems due to their advantages of precise targeting, high stability, safety, and improved immune system. Notably, algal-mediated nanoparticles (AMNPs), especially the microalgae-derived nanoparticles, are appealing as a sustainable delivery platform because of their biocompatibility and low toxicity in a homologous relationship. In addition, living microalgae demonstrated effective and regulated distribution into specified areas as the biohybrid microrobots. This review extensively summarised the uses of CRISPR/Cas systems in microalgae and the recent developments of nanoparticle-based CRISPR/Cas delivery systems. A systematic description of the properties and uses of AMNPs, microalgae-derived nanoparticles, and microalgae microrobots has also been discussed. Finally, this review highlights the challenges and future research directions for the development of gene-edited microalgae.
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