Schuierer, S. | bioRxiv Science

bioRxiv2019

A simple and efficient CRISPR technique for protein tagging

Genetic knock-in using homology directed repair is an inefficient process, requiring selection of few modified cells and hindering its application to primary cells. Here we describe Homology independent gene Tagging (HiTag), a method to tag a protein of interest by CRISPR in up to 66% transfected cells with one single electroporation. The technique has proven effective in various cell types, can be used to knock in a fluorescent protein for live cell imaging, to modify the cellular location of a target protein and to monitor levels of a protein of interest by a luciferase assay in primary cells.

cell biology↗

bioRxiv2019

CellSIUS provides sensitive and specific detection of rare cell populations from complex single cell RNA-seq data

Comprehensive benchmarking of computational methods for single-cell RNA sequencing (scRNA-seq) analysis is scarce. Using a modular workflow and a large dataset with known cell composition, we benchmarked feature selection and clustering methodologies for scRNA-seq data. Results highlighted a methodology gap for rare cell population identification for which we developed CellSIUS (Cell Subtype Identification from Upregulated gene Sets). CellSIUS outperformed existing approaches, enabled the identification of rare cell populations and, in contrast to other methods, simultaneously revealed transcriptomic signatures indicative of the rare cells function. We exemplified the use of our workflow and CellSIUS for the characterization of a human pluripotent cell 3D spheroid differentiation protocol recapitulating deep-layer corticogenesis in vitro. Results revealed lineage bifurcation between Cajal-Retzius cells and layer V/VI neurons as well as rare cell populations that differ by migratory, metabolic, or cell cycle status, including a choroid plexus neuroepithelial subgroup, revealing previously unrecognized complexity in human stem cell-derived cellular populations.

bioinformatics↗