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Found: 26 130
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DNA sequencing and CRISPR-Cas9 gene editing for target validation in mammalian cells.
Identification and validation of drug-resistant mutations can provide important insights into the mechanism of action of a compound. Here we demonstrate the feasibility of such an approach in mammalian cells using next-generation sequencing of drug-resistant clones and CRISPR-Cas9-mediated gene editing on two drug-target pairs, 6-thioguanine-HPRT1 and triptolide-ERCC3. We showed that disrupting functional HPRT1 allele or introducing ERCC3 point mutations by gene editing can confer drug resistance in cells.
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Improving gene targeting efficiency on pig IGF2 mediated by ZFNs and CRISPR/Cas9 by using SSA reporter system.
IGF2 (Insulin-like growth factor 2) is a major growth factor affecting porcine fetal and postnatal development. We propose that the precise modification of IGF2 gene of Chinese indigenous pig breed--Lantang pig by genome editing technology could reduce its backfat thickness, and increase its lean meat content. Here, we tested the genome editing activities of zinc finger nucleases (ZFNs) and CRISPR/Cas9 system on IGF2 gene in the Lantang porcine fetal fibroblasts (PEF). The results indicated that CRISPR/Cas9 presented cutting efficiency up to 9.2%, which was significantly higher than that generated by ZFNs with DNA cutting efficiency lower than 1%. However, even by using CRISPR/Cas9, the relatively lower percentage of genetically modified cells in the transfected population was not satisfied for somatic nuclear transfer (SCNT). Therefore, we used a SSA (Single-strand annealing) reporter system to enrich genetically modified cells induced by ZFN or CRISPR/Cas9. T7 endonuclease I assay revealed that this strategy improved genome editing activity of CRISPR/Cas9 by 5 folds, and was even more effective for improving genome editing efficiency of ZFN.
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Virus-induced genome editing enables functional genomics across diverse plant species.
Virus-induced genome editing (VIGE) is expanding plant functional genomics by enabling precise and heritable genome modification across diverse species. While classical model systems such as Arabidopsis thaliana have provided foundational genetic insights, many ecologically, agriculturally, and chemically important traits reside in species that remain difficult to manipulate genetically. By coupling CRISPR-Cas systems with plant viral vectors, VIGE bypasses key limitations of conventional transformation and enables rapid mutagenesis without repeated tissue culture and plant regeneration. This approach enables researchers to examine gene function in species selected for biological relevance rather than technical convenience. Here, we review the conceptual framework, technical considerations, and applications of VIGE, and discuss its current limitations and future prospects in ecological, evolutionary, and crop research.
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Small indels induced by CRISPR/Cas9 in the 5' region of microRNA lead to its depletion and Drosha processing retardance.
MicroRNA knockout by genome editing technologies is promising. In order to extend the application of the technology and to investigate the function of a specific miRNA, we used CRISPR/Cas9 to deplete human miR-93 from a cluster by targeting its 5' region in HeLa cells. Various small indels were induced in the targeted region containing the Drosha processing site and seed sequences. Interestingly, we found that even a single nucleotide deletion led to complete knockout of the target miRNA with high specificity. Functional knockout was confirmed by phenotype analysis. Furthermore, de novo microRNAs were not found by RNA-seq. Nevertheless, expression of the pri-microRNAs was increased. When combined with structural analysis, the data indicated that biogenesis was impaired. Altogether, we showed that small indels in the 5' region of a microRNA result in sequence depletion as well as Drosha processing retard.
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Application of CRISPR/Cas9 in Understanding Avian Viruses and Developing Poultry Vaccines.
Clustered regularly interspaced short palindromic repeats associated protein nuclease 9 (CRISPR-Cas9) technology offers novel approaches to precisely, cost-effectively, and user-friendly edit genomes for a wide array of applications and across multiple disciplines. This methodology can be leveraged to underpin host-virus interactions, elucidate viral gene functions, and to develop recombinant vaccines. The successful utilization of CRISPR/Cas9 in editing viral genomes has paved the way of developing novel and multiplex viral vectored poultry vaccines. Furthermore, CRISPR/Cas9 can be exploited to rectify major limitations of conventional approaches including reversion to virulent form, recombination with field viruses and transgene, and genome instability. This review provides comprehensive analysis of the potential of CRISPR/Cas9 genome editing technique in understanding avian virus-host interactions and developing novel poultry vaccines. Finally, we discuss the simplest and practical aspects of genome editing approaches in generating multivalent recombinant poultry vaccines that conform simultaneous protection against major avian diseases.
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Combining CRISPR/Cas9 and rAAV Templates for Efficient Gene Editing.
Altering endogenous genes in cells is an integral tool of modern cell biology. The ease-of-use of the CRISPR/Cas9 system to introduce genomic DNA breaks at specific sites in vivo has led to its rapid and wide adoption. In the absence of a DNA template, the lesion is repaired by nonhomologous end joining resolving as internal deletions. However, in the presence of a homologous DNA template, homology-directed repair occurs with variable efficiencies. Recent work has demonstrated that highly efficient gene targeting can be induced by combining CRISPR/Cas9 targeting of genomic loci with recombinant adeno-associated virus (rAAV) to provide a single-stranded homologous DNA template. Here we review the current state of CRISPR/Cas-based gene editing and provide a practical guide to applying the CRISPR/Cas and rAAV system for highly efficient, time- and cost-effective gene targeting.
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Using CRISPR-Cas9 to Study ERK Signaling in Drosophila.
Genome engineering using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated nuclease 9 (Cas9) technology is revolutionizing biomedical research. CRISPR-Cas9 enables precise editing of genes in a wide variety of cells and organisms, thereby accelerating molecular studies via targeted mutagenesis, epitope tagging, and other custom genetic modifications. Here, we illustrate the CRISPR-Cas9 methodology by focusing on Capicua (Cic), a nuclear transcriptional repressor directly phosphorylated and inactivated by ERK/MAPK. Specifically, we use CRISPR-Cas9 for targeting an ERK docking site of Drosophila Cic, thus generating ERK-insensitive mutants of this important signaling sensor.
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Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 with improved proof-reading enhances homology-directed repair.
Genome editing using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) predominantly induces non-homologous end joining (NHEJ), which generates random insertions or deletions, whereas homology-directed repair (HDR), which generates precise recombination products, is useful for wider applications. However, the factors that determine the ratio of HDR to NHEJ products after CRISPR/Cas9 editing remain unclear, and methods by which the proportion of HDR products can be increased have not yet been fully established. We systematically analyzed the HDR and NHEJ products after genome editing using various modified guide RNAs (gRNAs) and Cas9 variants with an enhanced conformational checkpoint to improve the fidelity at endogenous gene loci in HEK293T cells and HeLa cells. We found that these modified gRNAs and Cas9 variants were able to enhance HDR in both single-nucleotide substitutions and a multi-kb DNA fragment insertion. Our results suggest that the original CRISPR/Cas9 system from the bacterial immune system is not necessarily the best option for the induction of HDR in genome editing and indicate that the modulation of the kinetics of conformational checkpoints of Cas9 can optimize the HDR/NHEJ ratio.
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Recent Progress in Genome Editing for Gene Therapy Applications: The French Perspective.
Recent advances in genome editing tools, especially novel developments in the clustered regularly interspaced short palindromic repeats associated to Cas9 nucleases (CRISPR/Cas9)-derived editing machinery, have revolutionized not only basic science but, importantly, also the gene therapy field. Their flexibility and ability to introduce precise modifications in the genome to disrupt or correct genes or insert expression cassettes in safe harbors in the genome underline their potential applications as a medicine of the future to cure many genetic diseases. In this review, we give an overview of the recent progress made by French researchers in the field of therapeutic genome editing, while putting their work in the general context of advances made in the field. We focus on recent hematopoietic stem cell gene editing strategies for blood diseases affecting the red blood cells or blood coagulation as well as lysosomal storage diseases. We report on a genome editing-based therapy for muscular dystrophy and the potency of T cell gene editing to increase anticancer activity of chimeric antigen receptor T cells to combat cancer. We will also discuss technical obstacles and side effects such as unwanted editing activity that need to be surmounted on the way toward a clinical implementation of genome editing. We propose here improvements developed today, including by French researchers to overcome the editing-related genotoxicity and improve editing precision by the use of novel recombinant nuclease-based systems such as nickases, base editors, and prime editors. Finally, a solution is proposed to resolve the cellular toxicity induced by the systems employed for gene editing machinery delivery.
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RNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection.
AIDS remains incurable due to the permanent integration of HIV-1 into the host genome, imparting risk of viral reactivation even after antiretroviral therapy. New strategies are needed to ablate the viral genome from latently infected cells, because current methods are too inefficient and prone to adverse off-target effects. To eliminate the integrated HIV-1 genome, we used the Cas9/guide RNA (gRNA) system, in single and multiplex configurations. We identified highly specific targets within the HIV-1 LTR U3 region that were efficiently edited by Cas9/gRNA, inactivating viral gene expression and replication in latently infected microglial, promonocytic, and T cells. Cas9/gRNAs caused neither genotoxicity nor off-target editing to the host cells, and completely excised a 9,709-bp fragment of integrated proviral DNA that spanned from its 5' to 3' LTRs. Furthermore, the presence of multiplex gRNAs within Cas9-expressing cells prevented HIV-1 infection. Our results suggest that Cas9/gRNA can be engineered to provide a specific, efficacious prophylactic and therapeutic approach against AIDS.
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