Abstract
Human induced pluripotent stem (iPS) cell technologies coupled with genetic engineering now facilitate the study of the molecular underpinnings of disease in relevant human cell types. Application of CRISPR/Cas9-based approaches for genome-scale functional screening in iPS-derived cells, however, has been limited by technical constraints, including inefficient transduction in pooled format, loss of library representation, and poor cellular differentiation. Herein, we present optimized approaches for whole-genome CRISPR/Cas9 based screening in human iPS derived cardiomyocytes with near genome-wide representation at both the iPS and differentiated cell stages. As proof-of-concept, we perform a screen to investigate mechanisms underlying doxorubicin mediated cell death in iPS derived cardiomyocytes. We identified two poorly characterized, human-specific transporters (SLCO1A2, SLCO1B3) whose loss of function protects against doxorubicin-cardiotoxicity, but does not affect cell death in cancer cells. This study provides a technical framework for genome-wide functional screening in iPS derived cells and identifies new targets to mitigate doxorubicin-cardiotoxicity in humans.
Highlights
Human induced pluripotent stem cell technologies coupled with genetic engineering facilitate the study of the molecular underpinnings of disease in relevant human cell types
The discovery of human cellular plasticity and the reprogramming of adult somatic cells to induced pluripotent stem cells have ushered in new tools and approaches for interrogating human b iology1,2. iPS cells may be readily differentiated into a number of human cell t ypes[3,4,5], including terminally differentiated cells not typically amenable to isolation and culture, such as specialized neurons or c ardiomyocytes5–8. iPS derived cells have provided invaluable insights into biological processes and mechanisms underlying patient specific characteristics[9,10], cell type specific d ifferentiation[3,4,5], human disease s tates[11,12,13], and drug toxicity[14,15,16]
This study presents an accessible strategy for clustered regularly interspersed palindromic repeat (CRISPR)/Cas[9] mediated screening in human iPS derived cells at genome scale
Summary
Human induced pluripotent stem (iPS) cell technologies coupled with genetic engineering facilitate the study of the molecular underpinnings of disease in relevant human cell types. Application of CRISPR/Cas9-based approaches for genome-scale functional screening in iPS-derived cells, has been limited by technical constraints, including inefficient transduction in pooled format, loss of library representation, and poor cellular differentiation. This study provides a technical framework for genome-wide functional screening in iPS derived cells and identifies new targets to mitigate doxorubicin-cardiotoxicity in humans. Cardiomyocyte Differentiation iPSC Derived Cardiomyocytes d be determined whether infection with pooled lentiviral reagents required for genome-wide CRISPR sgRNA expression would alter either iPS cell stemness or differentiation capacity. It is unclear whether these approaches may be applied to reveal novel human-specific mechanisms underlying cardiomyocyte phenotypes
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