CasTuner: a degron and CRISPR/Cas-based toolkit for analog tuning of endogenous gene expression

Certain cellular processes are dose-dependent, requiring a specific quantity of gene products or a defined stoichiometry between them. This is exemplified by haploinsufficiency or by the need for dosage compensation for X-linked genes between the sexes in many species. Understanding dosage-sensitive processes requires the ability to perturb endogenous gene products in a quantitative manner. Here we present CasTuner, a CRISPR-based toolkit that allows analog tuning of endogenous gene expression. In the CasTuner system, activity of Cas-derived repressors is controlled through a FKBP12F36V degron domain and can thereby be quantitatively tuned by titrating the small molecule degrader dTAG-13. The toolkit can be applied at the transcriptional level, using the histone deacetylase hHDAC4 fused to dCas9, or at the post-transcriptional level, using the RNA-targeting CasRx. To optimise efficiency, inducibility and homogeneity of repression we target a fluorescently tagged endogenous gene, Esrrb, in mouse embryonic stem cells. Through flow cytometry, we show that CasTuner allows analog tuning of the target gene in a homogeneous manner across cells, as opposed to the widely used KRAB repressor domain, which exhibits a digital mode of action. We quantify repression and derepression dynamics for CasTuner and use it to measure dose-response curves between the pluripotency factor NANOG and several of its target genes, providing evidence for target-specific dose dependencies. CasTuner thus provides an easy-to-implement tool to perturb gene expression in an inducible, tunable and reversible manner and will be useful to study dose-responsive processes within their physiological contex

Advances of epigenetic editing

Epigenetic editing refers to the locus-specific targeting of epigenetic enzymes to rewrite the local epigenetic landscape of an endogenous genomic site, often with the aim of transcriptional reprogramming. Implementing clustered regularly interspaced short palindromic repeat-dCas9 greatly accelerated the advancement of epigenetic editing, yielding preclinical therapeutic successes using a variety of epigenetic enzymes. ,CRISPR/dCas9 Here, were review the current applications of these epigenetic editing tools in mammalians and shed light on biochemical improvements that facilitate versatile applications

Influence of medium composition on the characteristics of a denitrifying biofilm formed by alcaligenes denitrificans in a fluidised bed reactor

The influence of the ratio carbon/nitrogen and phosphorus concentration on the performance of a biofilm fluidised bed reactor used for denitrification and on the properties of the biofilm was studied. Although the removal efficiencies of C and N reached steady-state values, the thickness of the biofilm steadily increased. The dry density of the biofilm did not seem to be dependent on the loading conditions, although a denser biofilm was obtained when there was no nutrient limitation that corresponded to the complete removal of nitrate and carbon. The composition of the biofilm in terms of proteins and polysaccharides changed with the C/N ratio and P concentrations. Higher denitrifying activities, which were obtained with increasing P concentrations, were related with higher protein content, since metabolism was shifted from polysaccharide production towards cell production. The thickness and the density of the biofilms were related mainly with the shear stress in the reactor and the composition of biofilms was dependent on the composition of the medium and related with higher activities of the microorganisms.Fundação para a Ciência e a Tecnologia (FCT) - PRAXIS XXI . Instituto de Biotecnologia e Química Fina (IBQF)

Modifications of collagen and chromatin in ECM-related disease:Uncovering therapeutic targets for fibrosis and cancer

De extracellulaire matrix (ECM) is essentieel voor vele biologische functies en wordt strikt gereguleerd vanaf transcriptie tot post-translationeel niveau. Wanneer deze balans verstoord wordt kunnen er verscheidene ECM-gerelateerde ziekten ontstaan. Fibroproliferatieve aandoeningen, zoals weefselfibrose en tumoren, worden gekarakteriseerd door excessieve productie van het ECM eiwit collageen type I door myofibroblasten. Tevens, in onomkeerbare fibrose bevat collageen type I buitenproportioneel veel pyridinoline cross-links die de collageen fibrillen onafbreekbaar maken en zo weefselregeneratie in de weg zitten. Zodoende zijn fibroproliferatieve aandoeningen een enorme last voor de zorgsystemen wereldwijd en is er veel belang bij het ontwikkelen van nieuwe effectieve therapeutische behandelingen. De doelstelling van dit proefschrift was om meer inzicht te geven in het ontstaan van fibroproliferatieve aandoeningen, door meer te weten te komen over de moleculaire processen die betrokken zijn bij myofibroblast differentiatie, collageen biosynthese, en collageen cross-linken. Zo hebben we transcriptionele en post-translationele processen in kaart gebracht die van belang zijn voor het genereren van functioneel lysyl hydroxylase 2, het enzym dat zorg draagt voor pyridinoline cross-linking. Tevens identificeerden we een aantal chromatine-modificerende enzymen die de transcriptie beïnvloeden van genen die een rol spelen in diverse profibrotische processen. Als laatste hebben we biotechnologische tools ontwikkeld die we met succes hebben gebruikt om de transcriptionele activiteit van ziekte-gerelateerde genen langdurig aan te passen. De bevindingen in dit proefschrift zorgen niet alleen voor het beter begrijpen van fibrotische processen, maar bieden tevens nieuwe therapeutische mogelijkheden die van groot belang kunnen zijn voor het overkomen van ECM-gerelateerde ziekten

Molecular insights into prolyl and lysyl hydroxylation of fibrillar collagens in health and disease

Collagen is a macromolecule that has versatile roles in physiology, ranging from structural support to mediating cell signaling. Formation of mature collagen fibrils out of procollagen -chains requires a variety of enzymes and chaperones in a complex process spanning both intracellular and extracellular post-translational modifications. These processes include modifications of amino acids, folding of procollagen -chains into a triple-helical configuration and subsequent stabilization, facilitation of transportation out of the cell, cleavage of propeptides, aggregation, cross-link formation, and finally the formation of mature fibrils. Disruption of any of the proteins involved in these biosynthesis steps potentially result in a variety of connective tissue diseases because of a destabilized extracellular matrix. In this review, we give a revised overview of the enzymes and chaperones currently known to be relevant to the conversion of lysine and proline into hydroxyproline and hydroxylysine, respectively, and the O-glycosylation of hydroxylysine and give insights into the consequences when these steps are disrupted