Reactivating Fetal Hemoglobin Expression in Human Adult Erythroblasts Through BCL11A Knockdown Using Targeted Endonucleases.

We examined the efficiency, specificity, and mutational signatures of zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 systems designed to target the gene encoding the transcriptional repressor BCL11A, in human K562 cells and human CD34+ progenitor cells. ZFNs and TALENs were delivered as in vitro transcribed mRNA through electroporation; CRISPR/Cas9 was codelivered by Cas9 mRNA with plasmid-encoded guideRNA (gRNA) (pU6.g1) or in vitro transcribed gRNA (gR.1). Analyses of efficacy revealed that for these specific reagents and the delivery methods used, the ZFNs gave rise to more allelic disruption in the targeted locus compared to the TALENs and CRISPR/Cas9, which was associated with increased levels of fetal hemoglobin in erythroid cells produced in vitro from nuclease-treated CD34+ cells. Genome-wide analysis to evaluate the specificity of the nucleases revealed high specificity of this specific ZFN to the target site, while specific TALENs and CRISPRs evaluated showed off-target cleavage activity. ZFN gene-edited CD34+ cells had the capacity to engraft in NOD-PrkdcSCID-IL2Rγnull mice, while retaining multi-lineage potential, in contrast to TALEN gene-edited CD34+ cells. CRISPR engraftment levels mirrored the increased relative plasmid-mediated toxicity of pU6.g1/Cas9 in hematopoietic stem/progenitor cells (HSPCs), highlighting the value for the further improvements of CRISPR/Cas9 delivery in primary human HSPCs

Validation of national empirical pavement design approaches for cold recycled asphalt bases

The applied pavement design procedures differ from country to country within Europe. Especially the use of cold recycling materials which is handled similar to hot-mix asphalt (HMA) in the most available guidelines isn’t sufficient researched. To verify developed procedures, practical trial sections are indispensable. Within this study cold recycled asphalt base layers (CRAB) were assessed according to the German procedure for monitoring and evaluation of road surface conditions. It can be concluded, that the general applied empirical design approach, in which the CRAB layer thickness is increased by 50 % compared to a HMA base will result in a feasible pavement structure. In general, the re-designing of the sections according to various national empirical pavement design approaches partly results in high differences for same layer types

Religious Festivals Machines as Transition from Popular Culture\u2028Towards Industrial Design: Construction and Interpretation of the Giglio of Barra and Nola in Southern Italy

The aim of the paper is to describe, as an example of mediation between popular culture and industrial design, the case of a particular system of machines: those used in religious festivals of the Gigli in Barra and Nola, in Southern Italy. The Giglio is one of the four festival machines entered by UNESCO in 2013 in the intangible heritage of humanity list. It is a tower of wood, papier-mâché, nails and pins, 25m. high, that is carried on shoulder and "Ballad" with live music. The careful analysis of construction method of Giglio and the measured drawing of the structure, made it possible to understand the structural setting and construction mode for components. This mode represents a mediation between the concept of mass production and the single artefact, adhering to the formulation of a "standard dialect", as theorized in the early fifties of the last century

Reactivating Fetal Hemoglobin Expression in Human Adult Erythroblasts Through BCL11A Knockdown Using Targeted Endonucleases

We examined the efficiency, specificity, and mutational signatures of zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 systems designed to target the gene encoding the transcriptional repressor BCL11A, in human K562 cells and human CD34+ progenitor cells. ZFNs and TALENs were delivered as in vitro transcribed mRNA through electroporation; CRISPR/Cas9 was codelivered by Cas9 mRNA with plasmid-encoded guideRNA (gRNA) (pU6.g1) or in vitro transcribed gRNA (gR.1). Analyses of efficacy revealed that for these specific reagents and the delivery methods used, the ZFNs gave rise to more allelic disruption in the targeted locus compared to the TALENs and CRISPR/Cas9, which was associated with increased levels of fetal hemoglobin in erythroid cells produced in vitro from nuclease-treated CD34+ cells. Genome-wide analysis to evaluate the specificity of the nucleases revealed high specificity of this specific ZFN to the target site, while specific TALENs and CRISPRs evaluated showed off-target cleavage activity. ZFN gene-edited CD34+ cells had the capacity to engraft in NOD-PrkdcSCID-IL2Rγnull mice, while retaining multi-lineage potential, in contrast to TALEN gene-edited CD34+ cells. CRISPR engraftment levels mirrored the increased relative plasmid-mediated toxicity of pU6.g1/Cas9 in hematopoietic stem/progenitor cells (HSPCs), highlighting the value for the further improvements of CRISPR/Cas9 delivery in primary human HSPCs