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

Abstract

SummaryScaling 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.

A multiplexed gRNA piggyBac transposon system facilitates efficient induction of CRISPRi and CRISPRa in human pluripotent stem cells

“…The use of H1 was approved by Harvard University's ESCRO committee and methods were carried out in accordance with approved guidelines. Stem cells were grown in either mTeSR1 medium (Stem Cell Technologies 05850) or StemFlex medium (ThermoFisher A3349401) on Geltrex (Life Technologies A1413301) coated plates under conditions previously described 41 . Throughout culturing, cells were tested to confirm the absence of mycoplasma contamination (Lonza MycoAlert LT07-418).…”

CUB and Sushi Multiple Domains 1 (CSMD1) opposes the complement cascade in neural tissues

“…To investigate whether endogenous human CSMD1 can act as a complement inhibitor in human neural cells, we used a human stem cell line genetically edited to knock out CSMD1 (Hazelbaker et al, 2017). Stem cell-derived glutamatergic cortical-like neurons are one of the more well-characterized human neural cellular models, but it was unknown whether CSMD1 is expressed by glutamatergic cortical neurons in the human brain.…”

“…Step All embryonic stem cell work was performed according to an approved ESCRO protocol from Harvard University and the Broad Institute. CSMD1 KO and isogenic WT human embryonic stem cell lines (Hazelbaker et al, 2017) were differentiated using viral doxycycline-inducible overexpression of NGN2 (Nehme et al, 2018), co-infected with eGFP expressing virus, and cultured in 96-well, glass bottom plates (Senseo Greinier) for 14 days in vitro in serum-free medium without feeder cells. At day 14, a complement deposition assay was performed, modified from (Escudero-Esparza et al, 2013) to apply to cultured ES-derived neurons.…”

Multiplexed and Inducible Gene Modulation in Human Pluripotent Stem Cells by CRISPR Interference and Activation

“…The human embryonic stem cell line H1 (WA01) was obtained from WiCell Research Institute (Madison, WI) (Thomson et al, 1998). Stem cells were grown in either mTeSR1 medium (Stem Cell Technologies 05850) or StemFlex medium (ThermoFisher A3349401) on Geltrex (Life Technologies A1413301) coated plates under conditions previously described (Hazelbaker et al, 2017). Throughout culturing, cells were tested to confirm the absence of mycoplasma contamination (Lonza MycoAlert LT07-418).…”

“…Embryoid bodies (EBs) were generated as previously described (Hazelbaker et al, 2017). For immunostaining, hPSC colonies and EBs were fixed with 4% paraformaldehyde in PBS for 15 mins at room temperature (RT), blocked and permeabilized with 0.1% TritonX-100 and 4% serum in PBS for 1 hr at RT and incubated with the appropriate primary antibody at RT.…”