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Researchers Use Plasmid Gene Editing to Personalize Solid Tumor T-Cell Therapy.
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Lipopeptide-Based Nanosome-Mediated Delivery of Hyperaccurate CRISPR/Cas9 Ribonucleoprotein for Gene Editing.
A transient cytosolic delivery system for accurate Cas9 ribonucleoprotein is a key factor for target specificity of the CRIPSR/Cas9 toolkit. Owing to the large size of the Cas9 protein and a long negative strand RNA, the development of the delivery system is still a major challenge. Here, a size-controlled lipopeptide-based nanosome system is reported, derived from the blood-brain barrier-permeable dNP2 peptide which is capable of delivering a hyperaccurate Cas9 ribonucleoprotein complex (HypaRNP) into human cells for gene editing. Each nanosome is capable of encapsulating and delivering ≈2 HypaRNP molecules into the cytoplasm, followed by nuclear localization at 4 h post-treatment without significant cytotoxicity. The HypaRNP thus efficiently enacts endogenous eGFP silencing and editing in human embryonic kidney cells (up to 27.6%) and glioblastoma (up to 19.7% frequency of modification). The lipopeptide-based nanosome system shows superior delivery efficiency, high controllability, and simplicity, thus providing biocompatibility and versatile platform approach for CRISPR-mediated transient gene editing applications.
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UN could lead debate on gene editing.
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Efficient genome editing in a Mozambique tilapia cell line using CAS ribonucleoprotein complexes.
Genome editing using the CRISPR/Cas system makes it possible to rapidly characterise gene function in vitro and in vivo, and provides a powerful platform through which the genetics of farmed fish can be altered to improve traits such as resistance to important pathogens. Tilapia is one of the most important farmed fish globally; however, its farming is heavily impacted by Tilapia lake virus (TiLV). The Mozambique tilapia (Oreochromis mossambicus) brain (OmB) cell line is susceptible to TiLV, making it an ideal in vitro model for studying host-pathogen interactions and mechanisms of disease resistance. To establish OmB cells as a model for gene editing in Tilapia, it is essential to optimise genome editing protocols. In this study, we optimized a CRISPR/Cas9-based genome editing system for OmB cells using ribonucleoprotein complexes. With the optimized protocol, we successfully edited two endogenous genes with efficiencies ranging from 67% to 70%. In conclusion, we establish a highly efficient CRISPR/Cas9 RNP based gene editing workflow optimized for OmB tilapia cell lines. This optimized platform will facilitate future functional genomic studies in tilapia and support the development of TiLV-resistant tilapia.
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Advances and perspectives in the application of CRISPR/Cas9 in insects.
Insects compose more than half of all living organisms on earth, playing essential roles in global ecosystems and forming complex relationships with humans. Insect research has significant biological and practical importance. However, the application of genetic manipulation technology has long been restricted to several model insects only, such as gene knockout in Drosophila, which has severely restrained the development of insect biology research. Recently, with the increase in the release of insect genome data and the introduction of the CRISPR/Cas9 system for efficient genetic modification, it has been possible to conduct meaningful functional studies in a broad array of insect species. Here, we summarize the advances in CRISPR/Cas9 in different insect species, discuss methods for its promotion, and consider its application in future insect studies. This review provides detailed information about the application of the CRISPR/Cas9 system in insect research and presents possible ways to improve its use in functional studies and insect pest control.
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Reprogramming Mycobacterium tuberculosis CRISPR System for Gene Editing and Genome-wide RNA Interference Screening.
Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), which is still the leading cause of mortality from a single infectious disease worldwide. The development of novel anti-TB drugs and vaccines is severely hampered by the complicated and time-consuming genetic manipulation techniques for M. tuberculosis. Here, we harnessed an endogenous type III-A CRISPR/Cas10 system of M. tuberculosis for efficient gene editing and RNA interference (RNAi). This simple and easy method only needs to transform a single mini-CRISPR array plasmid, thus avoiding the introduction of exogenous protein and minimizing proteotoxicity. We demonstrated that M. tuberculosis genes can be efficiently and specifically knocked in/out by this system as confirmed by DNA high-throughput sequencing. This system was further applied to single- and multiple-gene RNAi. Moreover, we successfully performed genome-wide RNAi screening to identify M. tuberculosis genes regulating in vitro and intracellular growth. This system can be extensively used for exploring the functional genomics of M. tuberculosis and facilitate the development of novel anti-TB drugs and vaccines.
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Identification of Drug Resistance Genes Using a Pooled Lentiviral CRISPR/Cas9 Screening Approach.
In addition to advancing the development of gene-editing therapeutics, CRISPR/Cas9 is transforming how functional genetic studies are carried out in the lab. By increasing the ease with which genetic information can be inserted, deleted, or edited in cell and organism models, it facilitates genotype-phenotype analysis. Moreover, CRISPR/Cas9 has revolutionized the speed at which new genes underlying a particular phenotype can be identified through its application in genomic screens. Arrayed high-throughput and pooled lentiviral-based CRISPR/Cas9 screens have now been used in a wide variety of contexts, including the identification of essential genes, genes involved in cancer metastasis and tumor growth, and even genes involved in viral response. This technology has also been successfully used to identify drug targets and drug resistance mechanisms. Here, we provide a detailed protocol for performing a genome-wide pooled lentiviral CRISPR/Cas9 knockout screen to identify genetic modulators of a small-molecule drug. While we exemplify how to identify genes involved in resistance to a cytotoxic histone deacetylase inhibitor, Trichostatin A (TSA), the workflow we present can easily be adapted to different types of selections and other types of exogenous ligands or drugs.
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Applications of multiplexed CRISPR-Cas for genome engineering.
The CRISPR-Cas system has become a worldwide genome editing tool for various organisms. Its precision and efficiency have facilitated basic research, drug discovery and therapeutic interventions. In contrast to other genome editing agents, CRISPR-Cas is modulated by a short guide RNA. Due to its simplicity, CRISPR-Cas is recognized as the best candidate for multiplexed genome editing. With simultaneous targeting, efficient knockout of genes with large deletions is possible. In addition, CRISPR-Cas can induce complex structural variations, such as inversions, translocations and duplications. Moreover, by utilizing engineered CRISPR-Cas proteins specialized for direct repression or activation of gene expression, one can perform multiplexed epigenetic editing. Lastly, multiplexed targeting enables killing of specific types of cells by accumulating stress mediated by simultaneous DNA damages. Here we discuss how CRISPR-based editing technologies for multiple targets are applied in recent studies.
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Improving the justice-based argument for conducting human gene editing research to cure sickle cell disease.
In a recent article, Marilyn Baffoe-Bonnie offers three arguments that conducting CRISPR/Cas9 biotechnology research to cure sickle cell disease (SCD) would help address historical and current injustices in SCD research and care. I will grant that the first argument is sound, but show that the second and third arguments suffer from roughly the same defect, which is that they really argue for something else rather than for conducting CRISPR/Cas9 research to cure SCD. I conclude that a better justice-based argument would use only Baffoe-Bonnie's first argument.
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Clinical trials of germline gene editing: The exploitation problem.
The birth of the world's first genetically edited babies in 2018 provoked considerable ethical outrage. Nonetheless, many scientists and bioethicists now advocate the pursuit of clinical uses of human germline gene editing. Progress towards this goal will require research, including clinical trials where genetically edited embryos are implanted into a woman's uterus, gestated, and brought to term. This paper argues that such trials would likely conflict with the fundamental research ethical requirement of non-exploitation. This is because they would expose people who are in a vulnerable situation to risks and burdens that are substantial and not obviously offset by compensating benefits. I consider how the potential for exploitation in such trials might be mitigated, arguing that a feasible and justifiable approach is not easily found. If this analysis is correct, there is a significant ethical obstacle on the path towards clinical use of human germline gene editing.
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