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Stem Cell Reports. 2017 Oct 10;9(4):1315-1327. doi: 10.1016/j.stemcr.2017.09.006.

A Scaled Framework for CRISPR Editing of Human Pluripotent Stem Cells to Study Psychiatric Disease.

Author information

1
Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
2
Department of Pathology, Brigham and Women's Hospital, Boston, MA 02115, USA.
3
Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA. Electronic address: eggan@mcb.harvard.edu.
4
Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA. Electronic address: lbarrett@broadinstitute.org.

Abstract

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.

KEYWORDS:

CRISPR-Cas9; CRISPR-Cas9 nickase; human pluripotent stem cells; psychiatric disease; stem cell genomic stability; stem cell-derived human neurons; variant calling

PMID:
29020615
PMCID:
PMC5639480
DOI:
10.1016/j.stemcr.2017.09.006
[Indexed for MEDLINE]
Free PMC Article

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