575. High Content Analysis of CRISPR-Cas9 Gene-Edited Human Embryonic Stem Cells

Abstract

Gene-edited human cells are important resources for drug target identification, regulatory science, regenerative medicine and basic biology. Recently, use of the CRISPR-Cas9 system drastically cut the time required to produce gene-edited cell lines down to a few months. Targeted gene disruption in a population of human cells followed by selection and next-generation sequencing can identify drug targets, however many of these methods destroy all mutant clones, so a subsequent gene-editing experiment is required to obtain living mutant cells for downstream analysis. Overall, there is a need to increase the speed, multiplexing and precision in generating CRISPR-Cas9 mutants. ArrayEdit, a simple approach utilizing surface-modified multiwell plates containing one-pot transcribed single-guide RNAs (see FigureFigure), separates thousands of edited cell populations for automated, live, high-content imaging and analysis. The approach lowers the time and cost of gene editing and produces edited human embryonic stem cells at high efficiencies. Edited genes can be expressed in both pluripotent stem cells and differentiated cells from all three major germ layers. This allows for the real-time observation of mutations causing phenotypic differences that can be measured in in vitro tissues and organoids during culture as opposed to defined end points. The live cell and tissue assays on ArrayEdit are compatible with standard screening platforms and a variety of human cell types, thus permitting robust and sensitive detection of differences in proliferation, differentiation and other downstream effects of gene editing. This preclinical platform adds important capabilities to observe editing and selection in situ within complex structures generated by human cells, ultimately enabling optical and other molecular perturbations in the editing workflow that could refine the specificity and versatility of gene editing.FigureArrayEdit provides a new window into the process of gene editing human cells. Overview of ArrayEdit assembly and key components. Top: Schematic of one-pot PCR and T7 transcription. Bottom: Surface modification to the bottom of multiwell plates generates cell-adhesive µFeatures on a glass bottom. Each µFeature can be tracked over time via high-content imaging and stitched together to form a time-lapse visualization of edited cell phenotypes.View Large Image | Download PowerPoint Slide