techniki: CRISPR gene editing

Evidence: 100 • authority: 72

CRISPR gene editing (; pronounced like "crisper"; an abbreviation for "clustered regularly interspaced short palindromic repeats") is a genetic engineering technique in molecular biology by which the genomes of living organisms may be modified. It is based on a simplified version of the bacterial CRISPR-Cas9 antiviral defense system. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed or new ones added in vivo ("within the living"). The technique is considered highly significant in biotechnology and medicine as it enables in vivo genome editing and is considered exc…

Evidence: 49 • authority: 72

CRISPR (; acronym for clustered regularly interspaced short palindromic repeats) is a family of DNA sequences found in the genomes of prokaryotic organisms such as bacteria and archaea. Each sequence within an individual prokaryotic CRISPR is derived from a DNA fragment of a bacteriophage that had previously infected the prokaryote or one of its ancestors. These sequences are used to detect and destroy DNA from similar bacteriophages during subsequent infections. Hence these sequences play a key role in the antiviral (i.e. anti-phage) defense system of prokaryotes and provide a form of heritable, acquired immunity. CRISPR is found in approximately 50% of sequenced bacterial genomes and near…

Evidence: 45 • authority: 88

Endogenous intronic RNA tightly controls Cas9/CRISPR-mediated gene editing in human cells. ABSTRACT CRISPR/Cas approaches are often limited by off-target effects. In vivo, multiple cell types are often present and off-targets may result from unintended targeting of the wrong cells. In this work, we propose a solution to this limitation by using a transcribed intron of the target gene as an endogenous trigger (intron triggers) for a novel conditional guide RNA (intcgRNA). In vitro, intcgRNAs were responsive to the presence of the trigger. As a proof-of concept, the human IL2 receptor subunit gamma gene (IL2RG) was then targeted using both the intcgRNA and the corresponding conventional crRNA…

Evidence: 40 • authority: 72

CRISPR activation (CRISPRa) is a genetic perturbation technique that utilizes an engineered form of the CRISPR-Cas9 system to enhance the expression of specific genes without altering the underlying DNA sequence. Unlike traditional CRISPR-Cas9, which introduces double-strand breaks to edit genes, CRISPRa employs a modified, catalytically inactive Cas9 (dCas9) fused with transcriptional activators to target promoter or enhancer regions, thereby boosting gene transcription. This method allows for precise control of gene expression, making it a valuable tool for studying gene function, creating gene regulatory networks, and developing potential therapeutic interventions for a variety of diseas…

Evidence: 40 • authority: 80

5S rRNA Promoter for Guide RNA Expression Enabled Highly Efficient CRISPR/Cas9 Genome Editing in Aspergillus niger

Evidence: 21 • authority: 72

Genome editing, or genome engineering, or gene editing, is a type of genetic engineering in which DNA is inserted, deleted, modified or replaced in the genome of a living organism. Unlike early genetic engineering techniques that randomly insert genetic material into a host genome, genome editing targets the insertions to site-specific locations. Programmable molecules called restriction endonucleases, also known as gene editors or molecular scissors, are used to create double-strand breaks (DSBs) at desired locations in the genome. The induced breaks are repaired through homology-directed recombination (HDR) or non-homologous end joining (NHEJ), resulting in targeted mutations. The develop…

Evidence: — • authority: 88

Determining HIV-1 quasispecies genomic variability to optimize gRNA excision using the CRISPR/Cas9 gene editing system. In light of the global human immunodeficiency virus type 1 (HIV-1) epidemic, numerous attempts have been made to combat HIV-1 and cure patients of an otherwise deadly infection. While improving survival rates among HIV-1 infected patients, highly active retroviral therapy (HAART) and related therapies are at best a stalling measure due to the inevitable reemergence of escape virus from latently infected cellular reservoirs. Gene editing technologies hold the promise to permanently excise integrated virus from the genome. The general aim of this research was to thoroughly c…

Evidence: — • authority: 88

Virus-like particle-based delivery of Cas9/guide RNA ribonucleoprotein efficiently edits the brachyury gene and inhibits chordoma growth in vivo. Abstract Purpose Chordoma is a rare and aggressive bone cancer driven by the developmental transcription factor brachyury. Efforts to target brachyury are hampered by the absence of ligand-accessible small-molecule binding pockets. Genome editing with CRISPR systems provides an unprecedented opportunity to modulate undruggable transcription factor targets. However, delivery of CRISPR remains a bottleneck for in vivo therapy development. The aim was to investigate the in vivo therapeutic efficiency of Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) d…

Evidence: — • authority: 92

Highly frequent undesired insertional mutagenesis during Drosophila genome editing.. CRISPR/Cas9 based genome editing employing Homology Directed Repair (HDR) from template vector sequences is a widely used technique to enable precise insertions, deletions or modifications to genes. Here, we describe an undesired and highly frequent editing event when using conventional CRISPR/Cas9 plus HDR methods for Drosophila melanogaster germline genome editing. We find that the template vector employed for HDR repair unwantedly and commonly inserts into the genome. We observe this deviation from the desired edit at multiple genomic locations, with different HDR vectors and with multiple genome editing…

Evidence: — • authority: 92

Turnip Mosaic Virus-Based gRNA Delivery System for Plant Genome Editing.. Plant virus-based gRNA delivery systems offer a rapid alternative to stable transformation for CRISPR-mediated genome editing, but potyvirus-based platforms in Cas9-expressing plants are still underexplored. Here, we developed a turnip mosaic virus (TuMV)-based system for gRNA delivery in Cas9-expressing Nicotiana benthamiana and tested whether Csy4-mediated gRNA processing could improve editing efficiency. A TuMV construct carrying a gRNA targeting PHYTOENE DESATURASE ( NbPDS ) induced detectable editing in both infiltrated and systemic tissues, although editing frequencies were low. Incorporation of the bacterial en…

Evidence: — • authority: 92

Chemical and Structural Engineering of Guide RNAs for Precision Genome Editing: From Design Principles to Clinical Applications.. CRISPR-Cas9 has revolutionised genome editing by enabling efficient and programmable modification of defined DNA sequences, with guide RNAs (gRNAs) serving as indispensable elements that direct Cas9 to specific genomic loci. Initially regarded as auxiliary components, gRNAs are now recognized as critical determinants of editing efficiency and specificity and have attracted growing attention as independent targets for engineering. Chemical modification, sequence optimisation, and structural alteration of gRNAs have been shown to enhance on-target activity, suppres…

Evidence: — • authority: 92

An Electroporation-Based Protocol for Ex Vivo Base Editing: From Design to Quantitative Assessment.. Base editing is a CRISPR variant approach that enables single-nucleotide conversions without generating double-strand breaks. Cytosine and adenine base editors mediate C•G to T•A and A•T to G•C transitions, respectively, by coupling a deaminase to a catalytically impaired Cas9, a modified nuclease that lacks DNA cleavage activity but retains DNA binding capability. By avoiding double-strand breaks, base editing limits reliance on unpredictable end-joining pathways and facilitates more precise outcomes. This chapter provides a practical protocol for base editing by electroporation in adherent…