The Allen Cell Collection: Fluorescent Protein‐tagged Human Induced Pluripotent Stem Cell (hiPSC) Lines to Investigate Cell and Nuclear Organization in Differentiation, Disease, and Regeneration

Abstract

The Allen Institute for Cell Science is creating a collection of fluorescent protein‐tagged hiPS cell lines with the objective to illuminate cell organization and enable observation of cellular behavior from a structural perspective. To date, the Allen Cell Collection consists of >40 single‐, dual‐, or triple‐edited lines that have undergone extensive quality control testing to ensure genomic, cell biological, and stem cell integrity that are released and available for labs to obtain for their own research purposes. We have tagged the major cellular organelles, a few signaling molecules, membrane‐less and cardiomyocyte‐specific structures, phase transition markers, and transcription factors. Our most recently released lines include dual‐tagged mitochondria and microtubules (TOMM20/TUBA1B), and a telomere marker (TERF2). We have also released a dCas9 line (dCas9‐KRAB) which enables CRISPRi‐mediated knockdown of target genes and have leveraged this dCas9 edit to create a triple‐edited nucleolar line (FBL/NPM1/dCas9‐KRAB). Here, we present our gene‐editing and quality control methods for mono‐ and biallelic editing of expressed or silent genes that are expressed during differentiation. We also highlight the utility of our CRISPR interference hiPSC lines, which express dCas9‐KRAB, in a proof‐of‐concept, FACS‐based, pooled genetic screen, where we generated knockdowns that resulted in multiple nucleolar phenotypes, sorted cells by differing nucleolar morphology (condensed vs diffuse), and identified enriched CRISPR guides in each bin using Next‐Generation Sequencing (NGS). We now hypothesize that we can recapitulate these phenotypes with a targeted CRISPRi approach and imaging based on the hits we uncovered. Additionally, we have been testing a new gene editing approach to systematically tag new lines in our collection which utilizes adeno‐associated virus, serotype 6 (AAV6) in tandem with ribonucleoprotein transfection. Our hypothesis here is that our homology directed repair (HDR) efficiency will increase using AAV6 as compared to using plasmid donors. Initial trials have indicated higher efficiency, and we present results from these first experiments. Furthermore, we will present some of our openly available imaging tools such as the Allen Cell and Structure Segmenter to aid in your own imaging projects. Our cell lines, the plasmids used to generate them, thousands of segmented single cell 3D images of our lines, analysis and visualization tools, integrated cell models and biological findings are freely available to the research community (http://www.allencell.org/).