The development of novel genome editing tools has unlocked new opportunities that were not previously possible in basic and biomedical research. During the last two decades, several new genome editing methods have been developed that can be customized to modify specific regions of the genome. However, in the past couple of years, many newer and more exciting genome editing techniques have been developed that are more efficient, precise, and easier to use. These genome editing tools have helped to improve our understanding of genetic disorders by modeling them in cells and animal models, in addition to correcting the disease-causing mutations. Among the genome editing tools, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system has proven to be the most popular one due to its versatility and has been successfully used in a wide variety of laboratory animal models and plants. In this review, we summarize the customizable nucleases currently used for genome editing and their uses beyond the modification of genome. We also discuss the potential future applications of gene editing tools for both basic research and clinical purposes.
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http://dx.doi.org/10.3390/genes11090976 | DOI Listing |
Methods Mol Biol
January 2025
Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
CRISPR-Cas tools have recently been adapted for cell lineage tracing during development. Combined with single-cell RNA sequencing, these methods enable scalable lineage tracing with single-cell resolution. Here, I describe, scGESTALTv2, which combines cumulative CRISPR-Cas9 editing of a lineage barcode array with transcriptional profiling via droplet-based single-cell RNA sequencing (scRNA-seq).
View Article and Find Full Text PDFMethods Mol Biol
January 2025
Stem Cell Program, Boston Children's Hospital, Boston, MA, USA.
The CRISPR-activated repair lineage tracing (CARLIN) mouse line uses DNA barcoding to enable high-resolution tracing of cell lineages in vivo (Bowling et al, Cell 181, 1410-1422.e27, 2020). CARLIN mice contain expressed barcodes that allow simultaneous interrogation of lineage and gene expression information from single cells.
View Article and Find Full Text PDFMethods Mol Biol
January 2025
Charité Universitätsmedizin Berlin, Berlin, Germany.
A key goal of biology is to understand the origin of the many cell types that can be observed during diverse processes such as development, regeneration, and disease. Single-cell RNA-sequencing (scRNA-seq) is commonly used to identify cell types in a tissue or organ. However, organizing the resulting taxonomy of cell types into lineage trees to understand the origins of cell states and relationships between cells remains challenging.
View Article and Find Full Text PDFMethods Mol Biol
January 2025
Department of Plastic and Reconstructive Surgery, Johns Hopkins University, Baltimore, MD, USA.
FLP-FRT, a well-established technique for genome manipulation, and the revolutionary CRISPR/Cas9, known for its targeted indels, are combined in a novel approach. This unique method is applied to the Hox genes in the Drosophila melanogaster bithorax complex, which are closely located to the cis-regulatory modules that define their spatial-temporal regulation. The number and position of these genes are directly correlated to their expression pattern.
View Article and Find Full Text PDFTheranostics
January 2025
Laboratory of Molecular Genetics, College of Pharmacy, Chungbuk National University, Cheongju, 28160, Republic of Korea.
Cathepsin D (Ctsd) has emerged as a promising therapeutic target for Alzheimer's disease (AD) due to its role in degrading intracellular amyloid beta (Aβ). Enhancing Ctsd activity could reduce Aβ42 accumulation and restore the Aβ42/40 ratio, offering a potential AD treatment strategy. This study explored Ctsd demethylation in AD mouse models using dCas9-Tet1-mediated epigenome editing.
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