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Barrett, Lindy

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Barrett

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Lindy

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Barrett, Lindy
Author's Publications: search.filters.isAuthorOfPublication.7af40bf8-14d4-4028-994d-562240494942

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    Combining NGN2 Programming with Developmental Patterning Generates Human Excitatory Neurons with NMDAR-Mediated Synaptic Transmission
    (2018) Nehme, Ralda; Zuccaro, Emanuela; Dia Ghosh, Sulagna; Li, Chenchen; Sherwood, John; Pietilainen, Olli; Barrett, Lindy; Limone, Francesco; Worringer, Kathleen A.; Kommineni, Sravya; Zang, Ying; Cacchiarelli, Davide; Meissner, Alex; Adolfsson, Rolf; Haggarty, Stephen; Madison, Jon; Muller, Matthias; Arlotta, Paola; Fu, Zhanyan; Feng, Guoping; Eggan, Kevin
    SUMMARY Transcription factor programming of pluripotent stem cells (PSCs) has emerged as an approach to generate human neurons for disease modeling. However, programming schemes produce a variety of cell types, and those neurons that are made often retain an immature phenotype, which limits their utility in modeling neuronal processes, including synaptic transmission. We report that combining NGN2 programming with SMAD and WNT inhibition generates human patterned induced neurons (hpiNs). Single-cell analyses showed that hpiN cultures contained cells along a developmental continuum, ranging from poorly differentiated neuronal progenitors to well-differentiated, excitatory glutamatergic neurons. The most differentiated neurons could be identified using a CAMK2A::GFP reporter gene and exhibited greater functionality, including NMDAR-mediated synaptic transmission. We conclude that utilizing single-cell and reporter gene approaches for selecting successfully programmed cells for study will greatly enhance the utility of hpiNs and other programmed neuronal populations in the modeling of nervous system disorders.
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    A Scaled Framework for CRISPR Editing of Human Pluripotent Stem Cells to Study Psychiatric Disease
    (Elsevier, 2017) Hazelbaker, Dane; Beccard, Amanda; Bara, Anne M.; Dabkowski, Nicole; Messana, Angelica; Mazzucato, Patrizia; Lam, Daisy; Manning, Danielle; Eggan, Kevin; Barrett, Lindy
    Summary Scaling of CRISPR-Cas9 technology in human pluripotent stem cells (hPSCs) represents an important step for modeling complex disease and developing drug screens in human cells. However, variables affecting the scaling efficiency of gene editing in hPSCs remain poorly understood. Here, we report a standardized CRISPR-Cas9 approach, with robust benchmarking at each step, to successfully target and genotype a set of psychiatric disease-implicated genes in hPSCs and provide a resource of edited hPSC lines for six of these genes. We found that transcriptional state and nucleosome positioning around targeted loci was not correlated with editing efficiency. However, editing frequencies varied between different hPSC lines and correlated with genomic stability, underscoring the need for careful cell line selection and unbiased assessments of genomic integrity. Together, our step-by-step quantification and in-depth analyses provide an experimental roadmap for scaling Cas9-mediated editing in hPSCs to study psychiatric disease, with broader applicability for other polygenic diseases.