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: 26 079
Page 100 of 1000-
Simple and reliable detection of CRISPR-induced on-target effects by qgPCR and SNP genotyping.
The recent CRISPR revolution has provided researchers with powerful tools to perform genome editing in a variety of organisms. However, recent reports indicate widespread occurrence of unintended CRISPR-induced on-target effects (OnTEs) at the edited site in mice and human induced pluripotent stem cells (iPSCs) that escape standard quality controls. By altering gene expression of targeted or neighbouring genes, OnTEs can severely affect phenotypes of CRISPR-edited cells and organisms and thus lead to data misinterpretation, which can undermine the reliability of CRISPR-based studies. Here we describe a broadly applicable framework for detecting OnTEs in genome-edited cells and organisms after non-homologous end joining-mediated and homology-directed repair-mediated editing. Our protocol enables identification of OnTEs such as large deletions, large insertions, rearrangements or loss of heterozygosity (LOH). This is achieved by subjecting genomic DNA first to quantitative genotyping PCR (qgPCR), which determines the number of intact alleles at the target site using the same PCR amplicon that has been optimized for genotyping. This combination of genotyping and quantitation makes it possible to exclude clones with monoallelic OnTEs and hemizygous editing, which are often mischaracterized as correctly edited in standard Sanger sequencing. Second, occurrence of LOH around the edited locus is detected by genotyping neighbouring single-nucleotide polymorphisms (SNPs), using either a Sanger sequencing-based method or SNP microarrays. All steps are optimized to maximize simplicity and minimize cost to promote wide dissemination and applicability across the field. The entire protocol from genomic DNA extraction to OnTE exclusion can be performed in 6-9 d.
-
Selective loading and processing of prespacers for precise CRISPR adaptation.
CRISPR-Cas immunity protects prokaryotes against invading genetic elements1. It uses the highly conserved Cas1-Cas2 complex to establish inheritable memory (spacers)2-5. How Cas1-Cas2 acquires spacers from foreign DNA fragments (prespacers) and integrates them into the CRISPR locus in the correct orientation is unclear6,7. Here, using the high spatiotemporal resolution of single-molecule fluorescence, we show that Cas1-Cas2 selects precursors of prespacers from DNA in various forms-including single-stranded DNA and partial duplexes-in a manner that depends on both the length of the DNA strand and the presence of a protospacer adjacent motif (PAM) sequence. We also identify DnaQ exonucleases as enzymes that process the Cas1-Cas2-loaded prespacer precursors into mature prespacers of a suitable size for integration. Cas1-Cas2 protects the PAM sequence from maturation, which results in the production of asymmetrically trimmed prespacers and the subsequent integration of spacers in the correct orientation. Our results demonstrate the kinetic coordination of prespacer precursor selection and PAM trimming, providing insight into the mechanisms that underlie the integration of functional spacers in the CRISPR loci.
-
Editors' note.
-
RNA interference and CRISPR: Promising approaches to better understand and control citrus pathogens.
Citrus crops have great economic importance worldwide. However, citrus production faces many diseases caused by different pathogens, such as bacteria, oomycetes, fungi and viruses. To overcome important plant diseases in general, new technologies have been developed and applied to crop protection, including RNA interference (RNAi) and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) systems. RNAi has been demonstrated to be a powerful tool for application in plant defence mechanisms against different pathogens as well as their respective vectors, and CRISPR/Cas system has become widely used in gene editing or reprogramming or knocking out any chosen DNA/RNA sequence. In this article, we provide an overview of the use of RNAi and CRISPR/Cas technologies in management strategies to control several plants diseases, and we discuss how these strategies can be potentially used against citrus pathogens.
-
Leveraging CRISPR gene editing technology to optimize the efficacy, safety and accessibility of CAR T-cell therapy.
Chimeric Antigen Receptor (CAR)-T-cell therapy has revolutionized cancer immune therapy. However, challenges remain including increasing efficacy, reducing adverse events and increasing accessibility. Use of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology can effectively perform various functions such as precise integration, multi-gene editing, and genome-wide functional regulation. Additionally, CRISPR screening using large-scale guide RNA (gRNA) genetic perturbation provides an unbiased approach to understanding mechanisms underlying anti-cancer efficacy of CAR T-cells. Several emerging CRISPR tools with high specificity, controllability and efficiency are useful to modify CAR T-cells and identify new targets. In this review we summarize potential uses of the CRISPR system to improve results of CAR T-cells therapy including optimizing efficacy and safety and, developing universal CAR T-cells. We discuss challenges facing CRISPR gene editing and propose solutions highlighting future research directions in CAR T-cell therapy.
-
Synthetic multi-layer nanoparticles for CRISPR-Cas9 genome editing.
The clustered regularly interspaced short palindromic repeat (CRISPR) has great potential to revolutionize biomedical research and disease therapy. The specific and efficient genome editing strongly depends on high efficiency of delivery of the CRISPR payloads. However, optimization of CRISPR delivery vehicles still remains a major obstacle. Recently, various non-viral vectors have been utilized to deliver CRISPR tools. Many of these vectors have multi-layer structures assembled. In this review, we will introduce the development of CRISPR-Cas9 systems and their general therapeutic applications by summarizing current CRISPR-Cas9 based clinical trials. We will highlight the multi-layer nanoparticles (NPs) that have been developed to deliver CRISPR cargos in vitro and in vivo for various purposes, as well the potential building blocks of multi-layer NPs. We will also discuss the challenges in making the CRISPR tools into viable pharmaceutical products and provide potential solutions on efficiency and biosafety issues.
-
CRISPR/Cas9 Technology in Translational Biomedicine.
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) - RNA-guided Cas9 endonuclease system has provided a fast and efficient method for precise genome editing in diverse mammalian species, including humans. The CRISPR/Cas9 technology allows generation of modifications into site-specific locations of the selected genes in one major step by carrying deletions, insertions or DNA donor-directed precise sequence modifications. Cas9 forms a nucleoprotein complex with a sequence-specific guide RNA to create double-stranded breaks in complementary DNA target. Further, double-stranded break repair machinery leads to the intended gene modifications. The CRISPR/Cas9 system is widely used technique for genome modification, editing and other biotechnology applications, such as functional annotation, a system for visualization of specific genomic loci and transcriptional control of genes. CRISPR/Cas9-mediated manipulation of the laboratory animal genomes has contributed to the understanding of gene functions and has become a popular approach for modeling human disorders. Furthermore, the growing application of CRISPR-Cas9 system to human genes emerges as an extremely powerful technology for the molecular characterization and treatment of human disease. In this review we present the essential principles of CRISPR/Cas9 technology and the recent advances in its use in translational biomedicine.
-
Systematic identification of CRISPR off-target effects by CROss-seq.
-
Epitope recognition of magnetic peptide-imprinted chitosan composite nanoparticles for the extraction of CRISPR/dCas9a proteins from transfected cells.
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas9) technology is a powerful method for genetic modification (and regulation) that is of great current interest. The development of new, economical methods of detecting and extracting Cas9 (and/or dCas9) from transfected cells is thus an important advance. In this work, we employed molecular imprinting, using two peptides from the Cas9 protein, to make magnetic peptide-imprinted chitosan nanoparticles. dCas9 was encoded in a plasmid which was then transfected into human embryonic kidney (HEK293T) cells. The expression of dCas9 protein was measured by using total protein kits. Finally, the imprinted nanoparticles were used to extract dCas9 from transfected cell homogenates.
-
Single-Strand Annealing in Cancer.
DNA double-strand breaks (DSBs) are among the most serious forms of DNA damage. In humans, DSBs are repaired mainly by non-homologous end joining (NHEJ) and homologous recombination repair (HRR). Single-strand annealing (SSA), another DSB repair system, uses homologous repeats flanking a DSB to join DNA ends and is error-prone, as it removes DNA fragments between repeats along with one repeat. Many DNA deletions observed in cancer cells display homology at breakpoint junctions, suggesting the involvement of SSA. When multiple DSBs occur in different chromosomes, SSA may result in chromosomal translocations, essential in the pathogenesis of many cancers. Inhibition of RAD52 (RAD52 Homolog, DNA Repair Protein), the master regulator of SSA, results in decreased proliferation of BRCA1/2 (BRCA1/2 DNA Repair Associated)-deficient cells, occurring in many hereditary breast and ovarian cancer cases. Therefore, RAD52 may be targeted in synthetic lethality in cancer. SSA may modulate the response to platinum-based anticancer drugs and radiation. SSA may increase the efficacy of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR associated 9) genome editing and reduce its off-target effect. Several basic problems associated with SSA, including its evolutionary role, interplay with HRR and NHEJ and should be addressed to better understand its role in cancer pathogenesis and therapy.
PubMed returns only the first 10,000 results — refine the query to see the rest.