Project description:HMGN (high mobility group N) is a family of intrinsically disordered nuclear proteins that binds to nucleosomes, alters the structure of chromatin and affects transcription. A major unresolved question is the extent of functional specificity, or redundancy, between the various members of the HMGN protein family. Here we analyze the transcriptional profile of cells in which the expression of various HMGN proteins has been either deleted or doubled. The results reveal an HMGN-variant specific effect on the fidelity of the cellular transcription profile, indicating that functionally, the various HMGN subtypes are not fully redundant. RNA was collected from either primary knock-out MEFs or SV40-transformed MEFs and MIN6 cells over expressing various HMGN proteins and mutants and hybridized to Affymetrix arrays. We obtained a double ammount of HMGN proteins in MEFs and MIN6 cells by retroviral infection and subsequent selection procedure. We collected all infected cells (pools, not clones) in order to eliminate the effect of viral integration in the genome.
Project description:Auxin-inducible degron (AID) technology is powerful for chemogenetic control of proteolysis. However, generation of human cell lines to deplete endogenous proteins with AID remains challenging. Typically, homozygous degron-tagging efficiency is low and overexpression of an auxin receptor requires additional engineering steps. Here, we establish a one-step genome editing procedure with high-efficiency homozygous tagging and auxin receptor expression. We demonstrate its application in 5 human cell lines, including embryonic stem (ES) cells. The method allowed isolation of AID single-cell clones in 10 days for 11 target proteins with >80% average homozygous degron-tagging efficiency in A431 cells, and >50% efficiency for 5 targets in H9 ES cells. The tagged endogenous proteins were inducibly degraded in all cell lines, including ES cells and ES-cell derived neurons, with robust expected functional readouts. This method facilitates the application of AID for studying endogenous protein functions in human cells, especially in stem cells.
Project description:Targeted genome editing holds great promise in biology. However, efficient genome modification, including gene knock-in, remains an unattained goal in multiple cell types and loci due to poor transfection efficiencies and low target genes expression, impeding the positive selection of recombined cells. Here, we describe a genome editing approach to achieve efficient gene targeting using hard to transfect erythroid cell lines. We demonstrate robust fluorescent protein knock-in efficiency in low expressed transcription factor gene loci (e.g. Myb or Zeb1). We further show the ability to target several genes in individual cells, exemplified by MYB-GFP and NuMA-Cherry double knock-in, allowing multicolor labeling of regulatory factors at physiological endogenous levels. Our knock-in tagging approach allowed us to perform genome-wide TF analysis at increased signal-to-noise ratios, and highlighted previously unidentified MYB target genes and pathways. Overall, we establish a versatile CRISPR-Cas9-based platform, offering attractive opportunities for the dissection of the erythroid differentiation process.
Project description:In recent years, proximity labelling has established itself as an unbiased and powerful approach to map the interactome of specific proteins. While physiological expression of the labelling enzyme is beneficial for the mapping of interactors, generation of the desired cell lines remains time-consuming and challenging. Using our established pipeline for the rapid generation of C- and N-terminal CRISPR-Cas9 knock-ins (KIs) based on antibiotic selection, we were able to compare the performance of commonly used labelling enzymes when endogenously expressed. Endogenous tagging of the μ subunit of the AP-1 complex with TurboID allowed identification of known interactors and cargo proteins that simple overexpression of a labelling enzyme fusion protein could not reveal. We used the KI-strategy to compare the interactome of the different adaptor protein (AP) complexes and clathrin and were able to assemble lists of potential interactors and cargo proteins that are specific for each sorting pathway. Our approach greatly simplifies the execution of proximity labelling experiments for proteins in their native cellular environment and allows going from CRISPR transfection to mass spectrometry analysis and interactome data in just over a month.
Project description:We generated pluripotent stem cells (Mel1 hESC containing a GFP reporter driven by the endogenous insulin promoter) with a functional knock out of PTPN2 by CRISPR/Cas9 genome editing. KO or WT control stem cells were differentiated into beta-like cells (sBC), sorted for GFP, and prepared for deep sequencing.
Project description:At 35 DAP whole kernels (pericarp + endosperm + embryo) without glumes of green house grown ears of heterozygous (+/bt2-H2328), self-pollinated plants were visually divided into pools of phenotypically normal looking kernels (small indentation, slightly smaller than mutant kernels, genotype +/+ or +/bt2-H2328) and pools of phenotypically mutant kernels (plump, round kernels, slightly larger than normal kernels, genotype bt2-H2328/bt2-H2328). Pools consisted of 4 kernels. 3 different ears were used for a biological duplicate. Keywords: gene knock out
Project description:Identification of human deubiquitylating enzymes whose knock out result in hypersensitivity to DNA damaging agents, by comparing the sequence reads of 'barcode region' from mixed cell culture.