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

• Published in: Stem cell reports Vol. 9; no. 4; pp. 1315 - 1327
• Main Authors: Hazelbaker, Dane Z., Beccard, Amanda, Bara, Anne M., Dabkowski, Nicole, Messana, Angelica, Mazzucato, Patrizia, Lam, Daisy, Manning, Danielle, Eggan, Kevin, Barrett, Lindy E.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 10-10-2017; Elsevier
• Subjects: CRISPR-Cas9; CRISPR-Cas9 nickase; human pluripotent stem cells; psychiatric disease; Resource; stem cell genomic stability; stem cell-derived human neurons; variant calling; CRISPR-Cas9 nickase; stem cell-derived human neurons; stem cell genomic stability; CRISPR-Cas9; variant calling; human pluripotent stem cells; psychiatric disease
• ISSN: 2213-6711; 2213-6711
• DOI: 10.1016/j.stemcr.2017.09.006

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.
•The Cas9D10A-nickase strategy was effective in hPSCs across a diverse set of genes•Independent indel callers increased confidence in genotyping accuracy at scale•Editing efficiency varied between hPSC lines and correlated with genomic stability•Transcription state did not correlate with editing efficiency
In this article, Barrett, Eggan, and colleagues scale up a Cas9-mediated gene-targeting strategy in hPSCs to edit and genotype 58 distinct genes implicated in psychiatric disease. Their findings provide a roadmap for large-scale mutagenesis schemes as well as a set of validated sgRNAs, a resource of edited hPSC lines for six genes, and a novel indel analysis pipeline.