Your browser does not allow saving data (e.g. private mode): bookmarks will be lost when the tab closes.
Search PubMed
Keywords, author, journal, years — results from PubMed, abstracts with Russian translation, bookmarks in your browser.
Found: 26 233
Page 843 of 1000-
Shortening the Half-Life of Cas9 Maintains Its Gene Editing Ability and Reduces Neuronal Toxicity.
Virus-mediated expression of CRISPR/Cas9 is commonly used for genome editing in animal brains to model or treat neurological diseases, but the potential neurotoxicity of overexpressing bacterial Cas9 in the mammalian brain remains unknown. Through RNA sequencing (RNA-seq) analysis, we find that virus-mediated expression of Cas9 influences the expression of genes involved in neuronal functions. Reducing the half-life of Cas9 by tagging with geminin, whose expression is regulated by the cell cycle, maintains the genome editing capacity of Cas9 but significantly alleviates neurotoxicity. Thus, modification of Cas9 by shortening its half-life can help develop CRISPR/Cas9-based therapeutic approaches for treating neurological disorders.
-
Plant Epigenetic Editing to Analyze the Function of Histone Modifications in Gene-Specific Regulation.
Epigenetic editing enables the locus-specific manipulation of chromatin modifications. It allows the functional analysis of interactions between chromatin modifications and epigenetically stable gene expression states, thus establishing causal relationships, where previously correlations were suspected. Here, we describe the procedures for gene-specific epigenetic editing in plants that are based on targeting a histone modifier using an inactive dCas9 fusion protein that is recruited by a set of three distinct single guide RNAs (sgRNAs) that all target a region within the promoter of the target gene. We outline design principles and emphasize the need for suitable control constructs. In summary, the protocol will be widely useful for plant scientists looking to manipulate chromatin modifications in a locus-specific manner.
-
Choice of template delivery mitigates the genotoxic risk and adverse impact of editing in human hematopoietic stem cells.
Long-range gene editing by homology-directed repair (HDR) in hematopoietic stem/progenitor cells (HSPCs) often relies on viral transduction with recombinant adeno-associated viral vector (AAV) for template delivery. Here, we uncover unexpected load and prolonged persistence of AAV genomes and their fragments, which trigger sustained p53-mediated DNA damage response (DDR) upon recruiting the MRE11-RAD50-NBS1 (MRN) complex on the AAV inverted terminal repeats (ITRs). Accrual of viral DNA in cell-cycle-arrested HSPCs led to its frequent integration, predominantly in the form of transcriptionally competent ITRs, at nuclease on- and off-target sites. Optimized delivery of integrase-defective lentiviral vector (IDLV) induced lower DNA load and less persistent DDR, improving clonogenic capacity and editing efficiency in long-term repopulating HSPCs. Because insertions of viral DNA fragments are less frequent with IDLV, its choice for template delivery mitigates the adverse impact and genotoxic burden of HDR editing and should facilitate its clinical translation in HSPC gene therapy.
-
Predicting CRISPR/Cas9 Repair Outcomes by Attention-Based Deep Learning Framework.
As a simple and programmable nuclease-based genome editing tool, the CRISPR/Cas9 system has been widely used in target-gene repair and gene-expression regulation. The DNA mutation generated by CRISPR/Cas9-mediated double-strand breaks determines its biological and phenotypic effects. Experiments have demonstrated that CRISPR/Cas9-generated cellular-repair outcomes depend on local sequence features. Therefore, the repair outcomes after DNA break can be predicted by sequences near the cleavage sites. However, existing prediction methods rely on manually constructed features or insufficiently detailed prediction labels. They cannot satisfy clinical-level-prediction accuracy, which limit the performance of these models to existing knowledge about CRISPR/Cas9 editing. We predict 557 repair labels of DNA, covering the vast majority of Cas9-generated mutational outcomes, and build a deep learning model called Apindel, to predict CRISPR/Cas9 editing outcomes. Apindel, automatically, trains the sequence features of DNA with the GloVe model, introduces location information through Positional Encoding (PE), and embeds the trained-word vector matrixes into a deep learning model, containing BiLSTM and the Attention mechanism. Apindel has better performance and more detailed prediction categories than the most advanced DNA-mutation-predicting models. It, also, reveals that nucleotides at different positions relative to the cleavage sites have different influences on CRISPR/Cas9 editing outcomes.
-
Viability-Based Assessment Reveals True Efficiency of CRISPR-Cas9 Transfection Methods in Rabbit Spermatozoa.
Modern genome editing tools such as CRISPR-Cas9 have revolutionized mammalian genome engineering, yet translation to in vivo applications remains limited by low efficiency and frequent occurrence of mosaicism. Sperm-mediated delivery of editing reagents is one proposed alternative that may mitigate these issues. This method depends on efficient transfection of genome editing materials into viable spermatozoa, a critical yet frequently overlooked parameter. Using FACS, we compared electroporation (Neon NxT) and lipofection (CRISPRMAX) for introducing CRISPR-Cas9 ribonucleoproteins into viable rabbit spermatozoa. Electroporation, shown to enable Cas9 and plasmid transfection in spermatozoa from other species, performed poorly once dead spermatozoa were excluded. In contrast, Lipofectamine CRISPRMAX improved transfection efficiency with minimal effects on spermatozoa viability and motility. These findings emphasize the importance of distinguishing true transfection (transfection of viable spermatozoa) from total transfection and highlight lipofection as a promising alternative to electroporation for sperm-based genome editing, with potential applications in rabbit genome engineering.
-
Development and Characterization of a Factor V-Deficient CRISPR Cell Model for the Correction of Mutations.
Factor V deficiency, an ultra-rare congenital coagulopathy, is characterized by bleeding episodes that may be more or less intense as a function of the levels of coagulation factor activity present in plasma. Fresh-frozen plasma, often used to treat patients with factor V deficiency, is a scarcely effective palliative therapy with no specificity to the disease. CRISPR/Cas9-mediated gene editing, following precise deletion by non-homologous end-joining, has proven to be highly effective for modeling on a HepG2 cell line a mutation similar to the one detected in the factor V-deficient patient analyzed in this study, thus simulating the pathological phenotype. Additional CRISPR/Cas9-driven non-homologous end-joining precision deletion steps allowed correction of 41% of the factor V gene mutated cells, giving rise to a newly developed functional protein. Taking into account the plasma concentrations corresponding to the different levels of severity of factor V deficiency, it may be argued that the correction achieved in this study could, in ideal conditions, be sufficient to turn a severe phenotype into a mild or asymptomatic one.
-
Better living through chemistry: CRISPR/Cas engineered T cells for cancer immunotherapy.
T cells engineered to express transgenes such as chimeric antigen receptors (CAR) or modified T cell receptors (TCR) represent a new pillar of cancer therapy. Use of CRISPR/Cas gene-editing tools now allows even stronger and more precise control over the fate and function of engineered T cell therapies, including multiplex genome editing to facilitate use of off-the-shelf allogeneic T cells and novel approaches which have the potential to overcome some of the limitations of canonical Cas9-mediated DNA cleavage. This review summarizes the CRISPR/Cas techniques that have been used in preclinical research and outlines those that currently being tested in clinical trials.
-
Single-strand annealing: Molecular mechanisms and potential applications in CRISPR-Cas-based precision genome editing.
BACKGROUND: Spontaneous double-stranded DNA breaks (DSBs) frequently occur within the genome of all living organisms and must be well repaired for survival. Recently, more important roles of the DSB repair pathways that were previously thought to be minor pathways, such as single-strand annealing (SSA), have been shown. Nevertheless, the biochemical mechanisms and applications of the SSA pathway in genome editing have not been updated. PURPOSE AND SCOPE: Understanding the molecular mechanism of SSA is important to design potential applications in gene editing. This review provides insights into the recent progress of SSA studies and establishes a model for their potential applications in precision genome editing. SUMMARY AND CONCLUSION: The SSA mechanism involved in DNA DSB repair appears to be activated by a complex signaling cascade starting with broken end sensing and 5'-3' resection to reveal homologous repeats on the 3' ssDNA overhangs that flank the DSB. Annealing the repeats would help to amend the discontinuous ends and restore the intact genome, resulting in the missing of one repeat and the intervening sequence between the repeats. We proposed a model for CRISPR-Cas-based precision insertion or replacement of DNA fragments to take advantage of the characteristics. The proposed model can add a tool to extend the choice for precision gene editing. Nevertheless, the model needs to be experimentally validated and optimized with SSA-favorable conditions for practical applications.
-
History of genome editing: From meganucleases to CRISPR.
CRISPR-mediated genome editing has undoubtedly revolutionized genetic engineering of animals. With the ability for virtually unlimited modification of almost any genome it is easy to forget which amazing discoveries paved the way for this ground-breaking technology. Here, we summarize the history of genome editing platforms, starting from enhanced integration of foreign DNA by meganuclease-mediated double-strand breaks to CRISPR/Cas9, the leading technology to date, and its re-engineered variants.
-
Therapeutic genome editing with engineered nucleases.
Targeted genome editing with designer nucleases, such as zinc finger nucleases, TALE nucleases, and CRISPR-Cas nucleases, has heralded a new era in gene therapy. Genetic disorders, which have not been amenable to conventional gene-addition-type gene therapy approaches, such as disorders with dominant inheritance or diseases caused by mutations in tightly regulated genes, can now be treated by precise genome surgery. Moreover, engineered nucleases enable novel genetic interventions to fight infectious diseases or to improve cancer immunotherapies. Here, we review the development of the different classes of programmable nucleases, discuss the challenges and improvements in translating gene editing into clinical use, and give an outlook on what applications can expect to enter the clinic in the near future.
PubMed returns only the first 10,000 results — refine the query to see the rest.