Non-VLPs
Paul Pumpens in Single-Stranded RNA Phages, 2020
Concerning the immune activity of the CRISPR system against RNA phages, a breakthrough investigation was performed by Virginijus Šikšnys’ excellent team (Tamulaitis et al. 2014). By their investigation of nucleic acid specificity and mechanism of CRISPR interference for the Streptococcus thermophilus complex, the authors found that the type III-A StCsm complex targeted RNA and not DNA. When expressed in E. coli, the StCsm complex restricted the phage MS2 in a Csm3 nuclease-dependent manner. It was stressed by the authors that the phage MS2 is a preferable model to investigate RNA targeting by the CRISPR-Cas system in vivo because no DNA intermediate is formed during the life cycle of this phage. The pCRISPR_MS2 plasmid carried the synthetic CRISPR array of five repeats interspaced by four 36-nucleotide spacers targeting correspondingly the maturation, lysis, coat, and replicase sequences of MS2 RNA. The data demonstrated clearly that the StCsm complex conveyed in vivo resistance to the RNA phage MS2 in the heterologous E. coli host (Tamulaitis et al. 2014).
The CRISPR revolution and its potential impact on global health security
Published in Pathogens and Global Health, 2021
Kyle E. Watters, Jesse Kirkpatrick, Megan J. Palmer, Gregory D. Koblentz
CRISPR has been used to develop a suite of tools that scientists can use to better understand new and existing pathogens. One of the most beneficial uses of CRISPR for pathogen research has been the use of CRISPR interference to identify host factors critical to an agent’s replication cycle. With CRISPR interference, a catalytically dead Cas9 (dCas9) fused to a chromatin remodeling protein is guided to the beginning of a gene to silence expression [51]. By using a library of gRNAs covering the host organism’s genome, individual genes or gene clusters can be knocked down in parallel to identify which genes’ silencing leads to a survival phenotype [73]. Thus, in one straightforward experiment, a list of genes involved in pathogen biogenesis can be obtained. Similarly, CRISPR interference knockdowns can be used to work out the function of an agent’s genes without needing to make recombinant virus or create transgenic cell lines or animals, both of which can be time-consuming.
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].
SOD1-targeting therapies for neurodegenerative diseases: a review of current findings and future potential
Published in Expert Opinion on Orphan Drugs, 2020
John P. Franklin, Mimoun Azzouz, Pamela J. Shaw
A 2017 study by Gaj et al. described an AAV9-delivered, CRISPR/Cas9 system targeting the mutant SOD1 gene in SOD1G93A mice [105]. Here, neonatal mice were administered systemically with the AAV9-CRISPR/Cas9 as a one-off dose. Despite widespread delivery throughout the CNS, this resulted in very modest in vivo genome editing (0.2–0.4%) as measured by indel efficiency using deep sequencing. However, a striking 2.5-fold decrease in mutant SOD1 protein was seen in brain and spinal cord tissue. A 37% delay in disease onset, and a 25% increase in survival time were also observed in treated animals. The discrepancy between indel efficiency and clinical effect remains unaccounted for, but is a common occurrence in CRISPR-mediated genome editing, particularly in vivo. The authors postulate that CRISPR interference, a phenomenon whereby the Cas9 nuclease interrupts transcription but does not cleave, may play a role in explaining the difference [106]. They also noted that other in vivo knockdown studies using CRISPR/Cas9 have demonstrated a clinical effect that is disproportionate to the measured editing efficiency [105].
Related Knowledge Centers
- Archaea
- Bacteria
- Cas9
- Crispr
- Eukaryote
- Guide Rna
- Prokaryote
- Rna Interference
- Crispr Activation
- Transcription