Search results for "CRISPR"
showing 10 items of 68 documents
Cooperation between CRISPR-Cas types enables adaptation in an RNA-targeting system
2020
AbstractCRISPR-Cas immune systems adapt to new threats by acquiring spacers from invading nucleic acids such as phage genomes. However, some CRISPR-Cas loci lack genes necessary for spacer acquisition, despite apparent variation in spacer content between strains. It has been suggested that such loci may use acquisition machinery from co-occurring CRISPR-Cas systems. Here, using a lytic dsDNA phage, we observe spacer acquisition in the native host Flavobacterium columnare that carries an acquisition-deficient subtype VI-B locus and a complete subtype II-C locus. We characterize acquisition events in both loci and show that the RNA-targeting VI-B locus acquires spacers in trans using acquisit…
Comparison of CRISPR and marker based methods for the engineering of phage T7
2020
With the recent rise in interest in using lytic bacteriophages as therapeutic agents, there is an urgent requirement to understand their fundamental biology to enable the engineering of their genomes. Current methods of phage engineering rely on homologous recombination, followed by a system of selection to identify recombinant phages. For bacteriophage T7, the host genescmkortrxhave been used as a selection mechanism along with both type I and II CRISPR systems to select against wild-type phage and enrich for the desired mutant. Here we systematically compare all three systems; we show that the use of marker-based selection is the most efficient method and we use this to generate multiple …
2021
CRISPR-Cas immune systems adapt to new threats by acquiring new spacers from invading nucleic acids such as phage genomes. However, some CRISPR-Cas loci lack genes necessary for spacer acquisition despite variation in spacer content between microbial strains. It has been suggested that such loci may use acquisition machinery from cooccurring CRISPR-Cas systems within the same strain. Here, following infection by a virulent phage with a double-stranded DNA (dsDNA) genome, we observed spacer acquisition in the native host Flavobacterium columnare that carries an acquisition-deficient CRISPR-Cas subtype VI-B system and a complete subtype II-C system. We show that the VI-B locus acquires spacer…
Mucin induces CRISPR-Cas defence in an opportunistic pathogen
2021
AbstractParasitism by bacteriophages has led to the evolution of a variety of defense mechanisms in their host bacteria. However, it is unclear what factors lead to specific defenses being deployed upon phage infection. To explore this question, we exposed the bacterial fish pathogenFlavobacterium columnareto its virulent phage V156 in the presence of a eukaryotic host signal (mucin). All tested conditions led to some level of innate immunity, but the presence of mucin led to a dramatic increase in CRISPR spacer acquisition, especially in low nutrient conditions where over 60% of colonies had obtained at least one new spacer. Additionally, we show that the presence of a competitor bacterium…
2019
So far, studies on the bacterial immune system CRISPR-Cas and its ecological and evolutionary effects have been largely limited to laboratory conditions. While providing crucial information on the constituents of CRISPR-Cas, such studies may overlook fundamental components that affect bacterial immunity in natural habitats. Translating laboratory-derived predictions to nature is not a trivial task, owing partly to the instability of natural communities and difficulties in repeated sampling. To this end, we review how aquaculture, the farming of fishes and other aquatic species, may provide suitable semi-natural laboratories for examining the role of CRISPR-Cas in phage/bacterium coevolution…
Mucin induces CRISPR-Cas defense in an opportunistic pathogen
2022
It is unknown what circumstances promote particular bacterial defenses against bacterial viruses (phages). Almeida & Hoikkala et al. show that mucin, derived from mucus, greatly accelerates CRISPR-Cas defenses against phage in an opportunistic pathogen. Parasitism by bacteriophages has led to the evolution of a variety of defense mechanisms in their host bacteria. However, it is unclear what factors lead to specific defenses being deployed upon phage infection. To explore this question, we co-evolved the bacterial fish pathogen Flavobacterium columnare and its virulent phage V156 in presence and absence of a eukaryotic host signal (mucin) for sixteen weeks. The presence of mucin leads to a …
Modulação gênica em embriões humanos
2018
O recente implemento de moderna tecnologia -CRISPR/Cas9- possibilitou o avanço inquestionável da edição gênica em linhagem germinativa humana, seja por meio da manipulação de células precursoras de gametas, células tronco pluripotentes, gametas ou, até mesmo, de embriões. Mesmo levando em conta os benefícios terapêuticos preventivos demonstrados nas pesquisas básicas e préclínicas em embriões humanos, tais investigações encontram barreiras de aceitação em todo o mundo. Tal situação exige mudança de atitude no sentido de incrementar o debate em torno da aplicabilidade dessas novas tecnologias. Assim, a adequação das atuais regulamentações faz-se necessária e deve incluir a superação dos desa…
Genetic determinants of ATR inhibitor sensitivity and resistance in Gastric Cancer
2019
Synthetic lethal approaches in identifying genetic determinants of drug response is a powerful method in selecting patents for targeted cancer therapies. Ataxia-Telangiectasia Mutated (ATM) and Rad3-related protein kinase (ATR) is a valuable target to inhibit the DNA damage repair (DDR) pathway, that has been shown to be particularly effective in cancer cells harbouring other DDR defects, including truncating mutations in ARID1A, found in the 20% of gastric cancer (GC) patients. Although ATR inhibitors (ATRi) are emerging as promising cancer therapies, resistance mechanisms inevitably arise from these drugs as monotherapy, emphasising the importance of identifying genetic determinants of re…
Inactivation of peroxisomal ABC transporters, ABCD1 and ABCD2 in BV-2 microglial cells : Towards a better understanding of X-linked adrenoleukodystro…
2018
X-linked adrenoleukodystrophy (X-ALD) is a severe neurodegenerative disorder characterized by very-long-chain fatty acid (VLCFA) accumulation resulting from a peroxisomal β-oxidation defect. The disease is caused by mutations in the ABCD1 gene, which encodes for a peroxisomal half ABC transporter predicted, like its closest homologue ABCD2, to participate in the entry of VLCFA-CoA into the peroxisome, the unique site of their β-oxidation. Progress in understanding the physiopathogenesis of X-ALD suffers from the lack of appropriate cell and animal models. Since peroxisomal defects in microglia seem to be a key element of the onset of the disease, we generated four microglial cell lines unab…