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Approaches to Studying Polycystic Kidney Disease in Zebrafish
Published in Jinghua Hu, Yong Yu, Polycystic Kidney Disease, 2019
Recently, gene knockout technologies have developed very quickly. From zinc-finger nucleases (ZFNs), transcription activator-like effectors (TALENs), to the clustered regularly interspaced short palindromic repeats (CRISPR) system, all these different methods can generate gene-specific alterations via nonhomologous end-joining (NHEJ) by ZFN-, TALEN-, or CRISPR-associated-protein 9- (Cas9-) mediated double-stranded breaks. Among all these technologies, the CRISPR/Cas9 system is the most convenient and with high mutagenesis efficiency, has thus been widely used in the zebrafish field.21,22 Due to the high efficiency of gene knockout of the CRISPR/Cas9 system, it's possible to obtain phenotypic mutants in one generation or even right after injection, which saves 3–8 months (1–2 generation time) compared with classic genetic screen.23 By this method, it's easy to carry out a small-scale reverse genetic screen for candidate genes. Thus, it provides a powerful tool for genetic study in the zebrafish.
A Short Introduction to DNA Methylation
Published in Cristina Camprubí, Joan Blanco, Epigenetics and Assisted Reproduction, 2018
The functional relevance of DNA methylation (and other epigenetic changes) can now be interrogated by epigenetic editing using mainly a nuclease deficient version of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) 9 system, which allows recruiting chromatin modifying and remodeling complexes to specific target sequences (33). The fusion of the core catalytic domain of a DNA methyltransferase (e.g., DNMT3A) or demethylase (e.g., TET1) to a modified nuclease deficient Cas9 (dCas9) has been shown to induce specific targeted epigenetic changes either locally if a promoter is targeted or more regionally if a distant gene regulatory element such as an enhancer is targeted (33–35).
Nucleic Acids as Therapeutic Targets and Agents
Published in David E. Thurston, Ilona Pysz, Chemistry and Pharmacology of Anticancer Drugs, 2021
CRISPR-Cas9 is a gene-editing methodology that works by accurately cleaving a specific sequence of DNA within a given gene thus deleting it. Alternatively, a new piece of DNA can be inserted between the cut ends. The CRISPR-Cas9 acronym stands for “Clustered Regularly Interspaced Short Palindromic Repeats” and “CRISPR-Associated Protein 9”, respectively. CRISPR is a family of DNA sequences found within the genomes of prokaryotic organisms such as archaea and bacteria, originally derived from the DNA fragments of bacteriophages previously infecting the prokaryotes. They are used to detect and destroy DNA from similar phages during subsequent infections. Therefore, these sequences play a key role in the antiviral (i.e., antiphage) defense system of prokaryotes. In 2012, researchers Doudna and Emmanuelle Charpentier were the first to propose that the CRISPR-Cas9 system could be used for the programmable editing of genomes of any species including humans. It is now considered to be one of the most significant discoveries in the history of biology with potential as a powerful tool for both biomedical research and the development of novel treatments across multiple therapeutic areas including cancer. For research it is widely used in various fields for genetic manipulation due to its speed, high efficiency, and low cost. In the cancer area it has potential to be used to repair mutated genes known to be key to tumor progression (e.g., the mutated p53 tumor suppressor gene) or delete known oncogenes (e.g., BCL-2). Editing such genes at an early stage in cancer cells could reduce their ability to progress, and could also make them more sensitive to existing chemotherapy regimens and other treatments.
TYK2 as a novel therapeutic target in psoriasis
Published in Expert Review of Clinical Pharmacology, 2023
Sarah Elyoussfi, Shraddha S Rane, Steve Eyre, Richard B Warren
Experimental techniques include investigation of how transcription factors and other proteins interact with DNA (ChIP), chromatin structure and interactions (3C), and gene expression (eQTL and reporter gene assays) in the relevant cell types. Methods such as Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9 (CRISPR/Cas9) genome-editing systems can evaluate the effect of altering individual SNP alleles on gene expression [39]. By using relevant cell types, such as T-cells or keratinocytes, the effect of regulatory genome regions on subsequent gene expression can be examined using reporter gene assays. CRISPR interference systems can be used to downregulate or upregulate transcription of these regulatory regions. Informative functional readouts are then used to help us understand the function of disease-associated variants and the biological pathways on which they act [40].
Ex vivo gene therapy for lysosomal storage disorders: future perspectives
Published in Expert Opinion on Biological Therapy, 2023
Edina Poletto, Andrew Oliveira Silva, Ricardo Weinlich, Priscila Keiko Matsumoto Martin, Davi Coe Torres, Roberto Giugliani, Guilherme Baldo
Soon after the discovery of TALENs, a new gene editing tool was described from the adaptive immune system of bacteria against phages [21]. The easy design and lower cost of CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats- CRISPR associated protein 9) engineering, along with its high targeting efficiency across different cell types, has made it currently the most widely used gene editing tool in life sciences. It relies on a two-module system, in which the ectopic expression of a Cas endonuclease coupled to a guide RNA (gRNA) is able to recognize an enormous array of DNA sequences in the mammalian genome. Watson–Crick base pairing of the spacer sequence from the gRNA to its target DNA enables Cas nuclease activity, which promotes DSBs and triggers the same cellular DNA damage repair pathways described above. In addition to the spacer sequence, Cas effectors require the presence of a specific short sequence known as a protospacer-adjacent motif (PAM) near the target DNA site [22].
Hemophilia A gene therapy: current and next-generation approaches
Published in Expert Opinion on Biological Therapy, 2022
Steven W. Pipe, Gil Gonen-Yaacovi, Oscar G. Segurado
Targeted genome-editing techniques to correct gene mutations at the genome level using programmable nucleases (e.g. zinc-finger nuclease, transcription activator-like effector nuclease, and clustered regularly interspaced short palindromic repeat [CRISPR]/CRISPR-associated protein 9 [Cas9] systems [151]) may provide a more enduring treatment for hemophilia [102,152,153]. A zinc finger nuclease (ZFN)-based gene editing study was first explored in hemophilia B patients whereby it placed a normal F9 transgene within the albumin intron 1 under control of the endogeous albumin locus promoter. However, the program was terminated (NCT02695160). Recent findings showed that in vivo genome targeting of the human transgene into the Alb locus by CRISPR/Cas9 led to human FVIII production in the liver and ameliorated severe hemophilia A phenotype in mice [153]. Such genetic approaches effectively translated to humans may provide more permanent solutions to patients with hemophilia A.