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

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.

High content analysis platform for optimization of lipid mediated CRISPR-Cas9 delivery strategies in human cells

“…mCherry C sgRNA was selected based on initial screening of three sgRNAs targeting the mCherry locus as described in Section 2.2. Size (~100 nt) and purity of IVT sgRNA conformed to predictions based on the size of the template and were consistent with prior work [34]. Column purification of sgRNAs was necessary to remove unincorporated ribonucleotides prior to determining concentration.…”

“…Once designed, sgRNAs were synthesized as described previously [34]. Briefly, to construct template DNA for in vitro transcription (IVT), a 60 nt sgRNA specific forward primer, containing a truncated T7 promoter and the 20 nt target sequence, as well as a universal reverse primer were used to amplify a synthetic double stranded DNA template (Integrated DNA Technologies) encoding the conserved trans-activating CRISPR RNA (tracr) sequence.…”

“…Our platform exploits image cytometry and high content image analysis (HCA) in combination with a customized microcontact printed cell substrate [34,35] to simultaneously monitor non-viral delivery and editing in human cells. We use established image analysis methods [36] and design-of-experiments (DOE) based statistical techniques [37,38] to screen existing liposomal delivery materials and to optimize delivery inputs to maximize gene-editing outcomes.…”

Manufacturing Cell Therapies Using Engineered Biomaterials

“…Techniques such as micropatterning, that segregate populations into unique units, lend themselves well to high content imaging, analysis and screening [69] which enables facile assaying of phenotype and the selection of homogenous subpopulations. Previous studies demonstrated the utility of this in situ monitoring approach in evaluating heterogeneity in CRISPR-Cas9 delivery strategies [70] and editing of pluripotent stem cells [71]. Taken together, this body of work illustrates how biomaterials can provide a powerful platform for the guidance, monitoring, analysis, and quality control of cell populations.…”

Phenotypic screening with primary neurons to identify drug targets for regeneration and degeneration

“…CRISPR-Cas9 systems can be utilized in high-content/high-throughput screening to examine numerous effects; either performing focused screens or using a genome-wide gRNA library to enable genome-scale CRISPR-Cas9 knockout (GeCKO; Shalem et al, 2015). In this way, screens can examine changes in responses of cell cultures for drug discovery/resistance, bacterial toxin resistance, and validation of drug targets (Wang et al, 2014; Koike-Yusa et al, 2014; Zhou et al, 2014; Kasap et al, 2014; Shi et al, 2015; Smurnyy et al, 2014; Carlson-Stevermer et al, 2016; Hsu et al, 2014). Wei and colleagues demonstrated the advantages of high-throughput phenotypic screening based on the CRISPR/Cas9 system, utilizing single guide RNA (sgRNA) library transduced HeLa cells to identify novel genes related to anthrax and diphtheria toxin toxicity (Zhou et al, 2014)…”