Comparison of CRISPR/Cas endonucleases for in vivo retinal gene editing

CRISPR/Cas has opened the prospect of direct gene correction therapy for someinherited retinal diseases. Previous work has demonstrated the utility of adenoassociatedvirus (AAV) mediated delivery to retinal cells in vivo; however, with theexpanding repertoire of CRISPR/Cas endonucleases, it is not clear which of theseare most efficacious for retinal editing in vivo. We sought to compare CRISPR/Casendonuclease activity using both single and dual AAV delivery strategies for gene editingin retinal cells. Plasmids of a dual vector system with SpCas9, SaCas9, Cas12a, CjCas9and a sgRNA targeting YFP, as well as a single vector system with SaCas9/YFP sgRNAwere generated and validated in YFP-expressing HEK293A cell by flow cytometry andthe T7E1 assay. Paired CRISPR/Cas endonuclease and its best performing sgRNA wasthen packaged into an AAV2 capsid derivative, AAV7m8, and injected intravitreally intoCMV-Cre:Rosa26-YFP mice. SpCas9 and Cas12a achieved better knockout efficiencythan SaCas9 and CjCas9. Moreover, no significant difference in YFP gene editingwas found between single and dual CRISPR/SaCas9 vector systems. With a markedreduction of YFP-positive retinal cells, AAV7m8 delivered SpCas9 was found to havethe highest knockout efficacy among all investigated endonucleases. We demonstratethat the AAV7m8-mediated delivery of CRISPR/SpCas9 construct achieves the mostefficient gene modification in neurosensory retinal cells in vivo

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors

Repression of Global Protein Synthesis by Eif1a-Like Genes That Are Expressed Specifically in the Two-Cell Embryos and the Transient Zscan4-Positive State of Embryonic Stem Cells

Mouse embryonic stem (ES) cells are prototypical stem cells that remain undifferentiated in culture for long periods, yet maintain the ability to differentiate into essentially all cell types. Previously, we have reported that ES cells oscillate between two distinct states, which can be distinguished by the transient expression of Zscan4 genes originally identified for its specific expression in mouse two-cell stage embryos. Here, we report that the nascent protein synthesis is globally repressed in the Zscan4-positive state of ES cells, which is mediated by the transient expression of newly identified eukaryotic translation initiation factor 1A (Eif1a)-like genes. Eif1a-like genes, clustered on Chromosome 12, show the high sequence similarity to the Eifa1 and consist of 10 genes (Eif1al1-Eif1al10) and 9 pseudogenes (Eif1al-ps1-Eif1al-ps9). The analysis of the expressed sequence tag database showed that Eif1a-like genes are expressed mostly in the two-cell stage mouse embryos. Microarray analyses and quantitative real-time polymerase chain reaction analyses show that Eif1a-like genes are expressed specifically in the Zscan4-positive state of ES cells. These results indicate a novel mechanism to repress protein synthesis by Eif1a-like genes and a unique mode of protein synthesis regulation in ES cells, which undergo a transient and reversible repression of global protein synthesis in the Zscan4-positive state

Potentials of Cellular Reprogramming as a Novel Strategy for Neuroregeneration (vol 12, 460, 2018)

[This corrects the article DOI: 10.3389/fncel.2018.00460.]

Detection of SARS Coronavirus RNA in the Cerebrospinal Fluid of a Patient with Severe Acute Respiratory Syndrome

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Evaulating a low-intensity health education intervention on pneumococcal vaccination uptake among elders with chronic illinesses

Conference Theme: Cultivating Healthy Communitie

Le Courier de l'Europe

17 juillet 17781778/07/17 (VOL3,N5)-1778/07/17 (VOL4,N5).Appartient à l’ensemble documentaire : NordPdeC

Predictors of influenza vaccination in Chinese older patients with chronic disease in Hong Kong

Conference Theme: Asia-Pacific perspectives and evidence on health promotion and education: sharing experiences,efforts and evidenc