CRISPR/Cas9 technology can be used to investigate how double-strand breaks (DSBs) occurring in constitutive heterochromatin are getting repaired. This technology can be used to induce specific breaks on mouse pericentromeric heterochromatin, by using a guide RNA specific for the major satellite repeats and co-expressing it with Cas9. Those clean DSBs can be visualized later by confocal microscopy. More specifically, immunofluorescence can be used to visualize the main factors of each DSB repair pathway and quantify their percentage and pattern of recruitment at the heterochromatic region.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.1007/978-1-0716-0644-5_30 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Prevalent integration of genomic repetitive and regulatory elements and donor sequences at CRISPR-Cas9-induced breaks.
Commun Biol
January 2025
Bioscience Program, Biological and Environmental Science and Engineering Division, King Abdullah University of Science and Technology (KAUST), Thuwal, 23955-6900, Kingdom of Saudi Arabia.
CRISPR-Cas9 genome editing has been extensively applied in both academia and clinical settings, but its genotoxic risks, including large insertions (LgIns), remain poorly studied due to methodological limitations. This study presents the first detailed report of unintended LgIns consistently induced by different Cas9 editing regimes using various types of donors across multiple gene loci. Among these insertions, retrotransposable elements (REs) and host genomic coding and regulatory sequences are prevalent.
View Article and Find Full Text PDFStructural variations (SVs) play important roles in genetic diversity, evolution, and carcinogenesis and are, as such, important for human health. However, it remains unclear how spatial proximity of double-strand breaks (DSBs) affects the formation of SVs. To investigate if spatial proximity between two DSBs affects DNA repair, we used data from 3C experiments (Hi-C, ChIA-PET, and ChIP-seq) to identify highly interacting loci on six different chromosomes.
View Article and Find Full Text PDFComprehensive analysis of off-target and on-target effects resulting from liver-directed CRISPR-Cas9-mediated gene targeting with AAV vectors.
Mol Ther Methods Clin Dev
December 2024
Department of Gene Therapy & Regenerative Medicine, Vrije Universiteit Brussel (VUB), Brussels, Belgium.
Comprehensive genome-wide studies are needed to assess the consequences of adeno-associated virus (AAV) vector-mediated gene editing. We evaluated CRISPR-Cas-mediated on-target and off-target effects and examined the integration of the AAV vectors employed to deliver the CRISPR-Cas components to neonatal mice livers. The guide RNA (gRNA) was specifically designed to target the factor IX gene (F9).
View Article and Find Full Text PDFCRISPR/Cas9-induced double-strand breaks in the huntingtin locus lead to CAG repeat contraction through DNA end resection and homology-mediated repair.
BMC Biol
December 2024
Department of Genome Engineering, Institute of Bioorganic Chemistry, Polish Academy of Sciences, 61-704, Poznan, Poland.
Background: The expansion of CAG/CTG repeats in functionally unrelated genes is a causative factor in many inherited neurodegenerative disorders, including Huntington's disease (HD), spinocerebellar ataxias (SCAs), and myotonic dystrophy type 1 (DM1). Despite many years of research, the mechanism responsible for repeat instability is unknown, and recent findings indicate the key role of DNA repair in this process. The repair of DSBs induced by genome editing tools results in the shortening of long CAG/CTG repeats in yeast models.
View Article and Find Full Text PDFA p38 MAPK-ROS axis fuels proliferation stress and DNA damage during CRISPR-Cas9 gene editing in hematopoietic stem and progenitor cells.
Cell Rep Med
November 2024
San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, 20132 Milan, Italy; University School of Advanced Studies IUSS, 27100 Pavia, Italy. Electronic address:
Ex vivo activation is a prerequisite to reaching adequate levels of gene editing by homology-directed repair (HDR) for hematopoietic stem and progenitor cell (HSPC)-based clinical applications. Here, we show that shortening culture time mitigates the p53-mediated DNA damage response to CRISPR-Cas9-induced DNA double-strand breaks, enhancing the reconstitution capacity of edited HSPCs. However, this results in lower HDR efficiency, rendering ex vivo culture necessary yet detrimental.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!