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

Benjamin Steyer,Meng Lou,Arezoo Movaghar,Krishanu Saha,Ryan Prestil,Madelyn Goedland,Jared Carlson-Stevermer,Lucille Kohlenberg

doi:10.1016/j.stemcr.2015.11.014

Benjamin Steyer, Meng Lou + Show 6 more

Open Access

https://doi.org/10.1016/j.stemcr.2015.11.014

Copy DOI

Abstract

SummaryCRISPR-Cas9 gene editing of human cells and tissues holds much promise to advance medicine and biology, but standard editing methods require weeks to months of reagent preparation and selection where much or all of the initial edited samples are destroyed during analysis. ArrayEdit, a simple approach utilizing surface-modified multiwell plates containing one-pot transcribed single-guide RNAs, 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. 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.

Highlights

CRISPR-Cas9, an emerging genome surgery tool, exploits an engineered ribonucleoprotein complex consisting of two essential components: (1) a protein, Cas9; and (2) a single-guide RNA
CRISPR-Cas9 gene editing of human cells and tissues holds much promise to advance medicine and biology, but standard editing methods require weeks to months of reagent preparation and selection where much or all of the initial edited samples are destroyed during analysis
This preclinical platform adds important capabilities to observe editing and selection in situ within complex structures generated by human cells, enabling optical and other molecular perturbations in the editing workflow that could refine the specificity and versatility of gene editing

Summary

Introduction

CRISPR-Cas, an emerging genome surgery tool, exploits an engineered ribonucleoprotein complex consisting of two essential components: (1) a protein, Cas; and (2) a single-guide RNA (sgRNA). The Cas9-sgRNA complex cuts a specific target sequence in the genome. Human cells and tissues edited by CRISPR-Cas are important resources for drug target identification (Kasap et al, 2014; Shi et al, 2015; Smurnyy et al, 2014), regulatory science (Hsu et al, 2014), medicine (Doudna, 2015), and basic biology (Hsu et al, 2014; Sternberg and Doudna, 2015). There is a need to expand the throughput and capabilities of current in vitro human culture systems where novel genome surgery approaches can be evaluated with human cells and tissues (Baltimore et al, 2015). Advanced capabilities with human pluripotent stem cells in particular could eventually expand the suite of human preclinical model systems, ranging from patient-specific cell lines to complex human embryonic tissues established from stem cells

Results

Discussion

Conclusion