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CRISPR-Cas Gene Editing to the Genetic Rescue.
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Gene editing using CRISPR-Cas9 for the treatment of lung cancer.
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[From random mutagenesis to precise genome editing: the development and evolution of genome editing techniques in Drosophila].
Drosophila melanogaster, an important model organism for studying life science, has contributed more to the research of genetics, developmental biology and biomedicine with the development of genome editing techniques. Drosophila genome-editing techniques have evolved from random mutagenesis to precise genome editing and from simple mutant construction to diverse genome editing methods since the 20th century. Chemical mutagenesis, using Ethyl methanesulfonate (EMS), is an important technique to study gene function in forward genetics, however, the precise knockout of Drosophila genes could not be achieved. The gene targeting technology, based on homologous recombination, has accomplished the precise editing of Drosophila genome for the first time, but with low efficiency. The CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein)-mediated precise genome editing is simple, fast and highly efficient compared with the gene targeting technology in Drosophila. In this review, we focus on Drosophila gene knockout, and summarize the evolution of genome editing techniques in Drosophila, emphasizing the development and applications of gene targeting, zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) and CRISPR/Cas9 techniques.
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CRISPR-Cas9 Delivery by Artificial Virus (RRPHC).
Since its first harnessing in gene editing in 2012 and successful application in mammalian gene editing in 2013, the CRISPR-Cas9 system exerted magnificent power in all gene-editing-related applications, indicating a sharp thrive of this novel technology. However, there are still some critical drawbacks of the CRISPR-Cas9 system that hampered its broad application in gene editing. Efficient delivery of the Cas9 protein and its partner small guide RNA (sgRNA) to the target cells or tissue is one of the technical bottlenecks. CRISPR-Cas9 delivery via DNA plasmids still plays the big role in gene editing methods. With regard to the disadvantages of CRISPR-Cas9 plasmids, the most acute barrier lies in its large size (>10 kb) and the subsequent low transfection efficiency by conventional transfection method. In this chapter, what we present is an easy method by fabricating CRISPR-Cas9 plasmids into nanoparticle system and efficiently delivered into target cells to achieve gene editing.
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Genome Editing of Monkey.
Gene-modified monkey models would be particularly valuable in biomedical and neuroscience research. Virus-based transgenic and programmable nucleases-based site-specific gene editing methods (TALEN, CRISPR-cas9) enable the generation of gene-modified monkeys with gain or loss of function of specific genes. Here, we describe the generation of transgenic and knock-out (KO) monkeys with high efficiency by lentivirus and programmable nucleases.
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CRISPR/Cas9-Derived Mutations Both Inhibit HIV-1 Replication and Accelerate Viral Escape.
Cas9 cleaves specific DNA sequences with the assistance of a programmable single guide RNA (sgRNA). Repairing this broken DNA by the cell's error-prone non-homologous end joining (NHEJ) machinery leads to insertions and deletions (indels) that often impair DNA function. Using HIV-1, we have now demonstrated that many of these indels are indeed lethal for the virus, but that others lead to the emergence of replication competent viruses that are resistant to Cas9/sgRNA. This unexpected contribution of Cas9 to the development of viral resistance is facilitated by some indels that are not deleterious for viral replication, but that are refractory to recognition by the same sgRNA as a result of changing the target DNA sequences. This observation illustrates two opposite outcomes of Cas9/sgRNA action, i.e., inactivation of HIV-1 and acceleration of viral escape, thereby potentially limiting the use of Cas9/sgRNA in HIV-1 therapy.
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Efficient genome editing using endogenous U6 snRNA promoter-driven CRISPR/Cas9 sgRNA in Sclerotinia sclerotiorum.
We previously reported on a CRISPR-Cas9 genome editing system for the necrotrophic fungal plant pathogen Sclerotinia sclerotiorum. This system (the TrpC-sgRNA system), based on an RNA polymerase II (RNA Pol II) promoter (TrpC) to drive sgRNA transcription in vivo, was successful in creating gene insertion mutants. However, relatively low efficiency targeted gene editing hampered the application of this method for functional genomic research in S. sclerotiorum. To further optimize the CRISPR-Cas9 system, a plasmid-free Cas9 protein/sgRNA ribonucleoprotein (RNP)-mediated system (the RNP system) and a plasmid-based RNA polymerase III promoter (U6)-driven sgRNA transcription system (the U6-sgRNA system) were established and evaluated. The previously characterized oxaloacetate acetylhydrolase (Ssoah1) locus and a new locus encoding polyketide synthase12 (Sspks12) were targeted in this study to create loss-of-function mutants. The RNP system, similar to the TrpC-sgRNA system we previously reported, creates mutations at the Ssoah1 gene locus with comparable efficiency. However, neither system successfully generated mutations at the Sspks12 gene locus. The U6-sgRNA system exhibited a significantly higher efficiency of genemutation at both loci. This technology provides a simple and efficient strategy for targeted gene mutation and thereby will accelerating the pace of research of pathogenicity and development in this economically important plant pathogen.
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CRISPR cancer trial success paves the way for personalized treatments.
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Gene Editing and Genetic Lung Disease. Basic Research Meets Therapeutic Application.
Although our understanding of the genetics and pathology of congenital lung diseases such as surfactant protein deficiency, cystic fibrosis, and alpha-1 antitrypsin deficiency is extensive, treatment options are lacking. Because the lung is a barrier organ in direct communication with the external environment, targeted delivery of gene corrective technologies to the respiratory system via intratracheal or intranasal routes is an attractive option for therapy. CRISPR/Cas9 gene-editing technology is a promising approach to repairing or inactivating disease-causing mutations. Recent reports have provided proof of concept by using CRISPR/Cas9 to successfully repair or inactivate mutations in animal models of monogenic human diseases. Potential pulmonary applications of CRISPR/Cas9 gene editing include gene correction of monogenic diseases in pre- or postnatal lungs and ex vivo gene editing of patient-specific airway stem cells followed by autologous cell transplant. Strategies to enhance gene-editing efficiency and eliminate off-target effects by targeting pulmonary stem/progenitor cells and the assessment of short-term and long-term effects of gene editing are important considerations as the field advances. If methods continue to advance rapidly, CRISPR/Cas9-mediated gene editing may provide a novel opportunity to correct monogenic diseases of the respiratory system.
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Assaying Cell Cycle Progression via Flow Cytometry in CRISPR/Cas9-Treated Cells.
CRISPR/Cas9 system is a powerful technique for genome editing and engineering but obtaining a sizeable population of edited cells can be challenging for some cell types. CRISPR/Cas9-induced cell cycle arrest is a possible cause of this barrier to efficient editing; thus, it is desirable to know the cell cycle progression profile of any given cell line or type of interest resulting from CRISPR/Cas9 treatment. Here we describe a flow cytometry-based assay that enables the determination of cell cycle progression in the presence of CRISPR/Cas9 treatment, in addition to the transfection and expression efficiencies of Cas9 vectors. This assay can also easily determine the effect of various interventions on obtaining a larger pool of Cas9-treated cells.
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