RESUMO
Gene editing by CRISPR/Cas9 is commonly used to generate germline mutations or perform in vitro screens, but applicability for in vivo screening has so far been limited. Recently, it was shown that in Drosophila, Cas9 expression could be limited to a desired group of cells, allowing tissue-specific mutagenesis. Here, we thoroughly characterize tissue-specific (ts)CRISPR within the complex neuronal system of the Drosophila mushroom body. We report the generation of a library of gRNA-expressing plasmids and fly lines using optimized tools, which provides a valuable resource to the fly community. We demonstrate the application of our library in a large-scale in vivo screen, which reveals insights into developmental neuronal remodeling.
Assuntos
Animais Geneticamente Modificados/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Drosophila/genética , Edição de Genes/métodos , Animais , Sistemas CRISPR-Cas/genética , Feminino , Masculino , Corpos Pedunculados/metabolismo , Mutagênese , Sistema Nervoso/crescimento & desenvolvimento , Plasticidade Neuronal/genética , Neurônios/fisiologia , Plasmídeos/genética , RNA Guia de Cinetoplastídeos/genéticaRESUMO
BACKGROUND AND AIMS: The optimum serological test for celiac disease (CD) in young children is not known. The objective of our study was to compare the performance of three serological tests (IgA + IgG DGP, IgA TTG, and IgA + IgG EMA) for children younger than 3 years of age. METHODS: We identified all subjects younger than 3 years of age (n = 6,074) that were tested for CD serology and included those with biopsy data. Patients were classified as group 1 (n = 47): patients with confirmed CD or group 2 (n = 12): patients with normal biopsy findings. RESULTS: There was statistically significant difference between group 1 and group 2 with regard to number of patients with positive IgA TTG (97.87% vs. 50%, P < 0.001), IgA + IgG DGP (100% vs. 77.78%, P = 0.007), and IgA + IgG EMA (95.65% vs. 9.09%, P < 0.001). There was a significantly positive correlation between Marsh-Oberhuber score on the small duodenal biopsies and all tests. Analysis of sensitivity and specificity showed that manufacturer's levels had high sensitivity for all tests (IgA TTG 97%, IgA + IgG DGP 100%, IgA + IgG EMA 96%), however specificity was low for IgA + IgG DGP (44%) and IgA TTG (50%) but not for IgA + IgG EMA (91%). CONCLUSIONS: For children younger than 3 years of age, IgA + IgG EMA is highly sensitive and specific. Use of IgA + IgG DGP or IgA TTG as a single serological marker is insufficient for definite diagnosis of CD in this age group. Based on our results, it might be reasonable to postpone the biopsy for asymptomatic children with negative EMA.
