CRISPR-Cas9 systems provide a platform for high efficiency genome editing that are enabling innovative applications of mammalian cell engineering. However, the delivery of Cas9 and synthesis of guide RNA (gRNA) remain as steps that can limit overall efficiency and ease of use. Here we describe methods for rapid synthesis of gRNA and for delivery of Cas9 protein/gRNA ribonucleoprotein complexes (Cas9 RNPs) into a variety of mammalian cells through liposome-mediated transfection or electroporation. Using these methods, we report nuclease-mediated indel rates of up to 94% in Jurkat T cells and 87% in induced pluripotent stem cells (iPSC) for a single target. When we used this approach for multigene targeting in Jurkat cells we found that two-locus and three-locus indels were achieved in approximately 93% and 65% of the resulting isolated cell lines, respectively. Further, we found that the off-target cleavage rate is reduced using Cas9 protein when compared to plasmid DNA transfection. Taken together, we present a streamlined cell engineering workflow that enables gRNA design to analysis of edited cells in as little as four days and results in highly efficient genome modulation in hard-to-transfect cells. The reagent preparation and delivery to cells is amenable to high throughput, multiplexed genome-wide cell engineering.

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Induced pluripotent stem cells (iPSCs) are promising tools for disease research and cell therapy. One of the critical steps in establishing iPSC lines is the early identification of fully reprogrammed colonies among unreprogrammed fibroblasts and partially reprogrammed intermediates. Currently, colony morphology and pluripotent stem cell surface markers are used to identify iPSC colonies. Through additional clonal characterization, we show that these tools fail to distinguish partially reprogrammed intermediates from fully reprogrammed iPSCs. Thus, they can lead to the selection of suboptimal clones for expansion. A subsequent global transcriptome analysis revealed that the cell adhesion protein CD44 is a marker that differentiates between partially and fully reprogrammed cells. Immunohistochemistry and flow cytometry confirmed that CD44 is highly expressed in the human parental fibroblasts used for the reprogramming experiments. It is gradually lost throughout the reprogramming process and is absent in fully established iPSCs. When used in conjunction with pluripotent cell markers, CD44 staining results in the clear identification of fully reprogrammed cells. This combination of positive and negative surface markers allows for easier and more accurate iPSC detection and selection, thus reducing the effort spent on suboptimal iPSC clones.

Rapid technological developments for the efficient generation of footprint-free induced pluripotent stem cells (iPSC) enabled the creation of patient-specific iPSC for downstream applications in drug discovery and regenerative medicine. However, the large number of iPSCs, generated from diverse genetic backgrounds using various methods and culture conditions, created a steep challenge for rapid characterization and a demand for standardized methods. Current methods rely on a combination of in vitro and in vivo cellular analyses based on the expression of markers of self-renewal and the ability of the cells to differentiate into cell types representative of the three germ layers as a confirmation of functional pluripotency. These methods, though informative and extensively used, are not ideal for parallel analyses of large numbers of samples and hence not amenable to high-throughput environments. Recently, genetic and epigenetic expression signatures were used to define and confirm cell states, thus providing a surrogate molecular assay that can potentially replace complex in vivo cellular assays such as teratoma formation. In this chapter, we describe a molecular assay for rapid characterization and standardization of pluripotent stem cells. The TaqMan(®) hPSC Scorecard™ Panel is a comprehensive gene expression real-time PCR assay that consists of 94 individual q-PCR assays comprised of a combination of control, housekeeping, self-renewal, and lineage-specific genes. The resulting expression data set is analyzed using cloud-based analysis software that compares the expression pattern against a reference standard composed of multiple functionally validated ESC and iPSC lines. This system was successfully used to test several ESC and iPSC lines in their undifferentiated states to confirm their signatures of self renewal, as well as their terminally differentiates states, via spontaneous differentiation and directed differentiation into specific lineages, to determine the lines' differentiation potential. This genetic analysis tool, together with the flexibility to utilize varying sample inputs and preparation methods, provides a rapid method to confirm functional pluripotency of ESCs and iPSCs.

Ustilago maydis, a Basidiomycete fungus that infects maize, exhibits two basic morphologies, a yeast-like and a filamentous form. The yeast-like cell is elongated, divides by budding, and the bud grows by tip extension. The filamentous form divides at the apical cell and grows by tip extension. The repertoire of morphologies is increased during interaction with its host, suggesting that plant signals play an important role in generation of additional morphologies. We have used S. cerevisiae and S. pombe genes known to play a role in cell polarity and morphogenesis, and in the cytoskeleton as probes to survey the U. maydis genome. We have found that most of the yeast machinery is conserved in U. maydis, albeit the degree of similarity varies from strong to weak. The U. maydis genome contains the machinery for recognition and interpretation of the budding yeast axial and bipolar landmarks; however, genes coding for some of the landmark proteins are absent. Genes coding for cell polarity establishment, exocytosis, actin and microtubule organization, microtubule plus-end associated proteins, kinesins, and myosins are also present. Genes not present in Saccharomyces cerevisiae and Schizosaccharomyces pombe include a homologue of mammalian Rac, a hybrid myosin-chitin synthase, and several kinesins that exhibit more similarity to their mammalian counterparts. We also used the U. maydis genes identified in this analysis to search other fungal and other eukaryotic genomes to identify the closest homologues. In most cases, not surprisingly, the closest homologue is among filamentous fungi, not the yeasts, and in some cases it is among mammals.