Project description:C3 glomerulopathy (C3G) is a rare disease resulting from dysregulation of the alternative pathway (AP) of complement. C3G includes C3 glomerulonephritis (C3GN) and dense deposit disease (DDD). Both C3GN and DDD are characterized by bright glomerular C3 staining on immunofluorescence studies. However, on electron microscopy, DDD is characterized by dense osmiophilic mesangial and intramembranous deposits along the glomerular basement membranes (GBM), while the deposits of C3GN are not dense. Why the deposits appear dense in DDD and not in C3GN is not known.We performed laser microdissection of glomeruli followed by mass spectrometry in 12 known cases of DDD and C3GN each to determine the proteomic profile and differences between C3GN and DDD.
Project description:To further determine the sequence features of active cardiac enhancers, 123 cardiac enhancers were functionally dissected by 10 bp tiling deletion-based mutagenesis MPRA in iPSC-CMs.
Project description:Systematic comparison of CRISPR-based transcriptional activators uncovers gene-regulatory features of enhancer-promoter interactions
Project description:The CRISPR-Cas9 system enables efficient sequence-specific mutagenesis for creating germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-guideRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we established in vitro-assembled, fluorescent Cas9-sgRNA RNPs in stabilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. Sequence analysis of targeted loci in individual embryos reveals highly efficient bi-allelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis reveals preliminary loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show efficient targeting of functional non-coding elements in gene-regulatory regions using saturating mutagenesis towards uncovering functional control elements in transgenic reporters and endogenous genes. Our results suggest that in vitro assembled, fluorescent Cas9-sgRNA RNPs provide a rapid reverse-genetics tool for direct and scalable loss-of-function studies beyond zebrafish applications.