Genome-wide CRISPR/Cas9 screening in human iPS derived cardiomyocytes uncovers novel mediators of doxorubicin cardiotoxicity

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.

Figure 1

Optimization of CRISPR/Cas9 transfer in human iPS cells. (a) Schematic for forward genetic CRISPR/Cas9 screening in induced pluripotent stem (iPS) derived cardiomyocytes. (b) Optimization of infection efficiency with mCherry expressing virus. Cells were treated with no virus (blank), standard infection protocols (control), addition of 1% BSA during standard infection (+ BSA), removal of the ROCK inhibitor thiazovivin during plating (− Thiaz), plating at half density prior to standard infection (low density) or use of reverse transduction (n = 2 per condition, student’s T-test, **: p < 0.01). (c) Infection efficiency in iPS cells using standard (control) or reverse transduction approaches (n = 2 per condition). (d) Bright-field image of iPS cells during plating (top) and 24 h after plating (bottom).

Figure2

Phenotypic resilience in pooled CRISPR/Cas9 library infected iPS cells. Uninfected iPS cells (control) or those transduced with CRISPR/Cas9 library (virus) were assessed for (a) gene expression of pluripotency markers (n = 3 per condition), (b) proliferative potential (n = 3 per condition), and (c) cardiac troponin T (cTNT) protein expression after differentiation to cardiomyocytes. (d) Percent of CRISPR/Cas9 library sgRNAs present in iPS cells at 10 read-counts or higher. (e) Number of sgRNAs per gene present in plasmid, iPS cells, and iPS derived cardiomyocytes.

Figure 3

Genome-scale CRISPR/Cas9 screen in iPS derived cardiomyocytes for mediators of doxorubicin-toxicity. (a) Dose response curve for doxorubicin-toxicity in iPS derived cardiomyocytes (circulating concentration range highlighted in red, n = 8 per condition). (b) Distribution of fold change scores in doxorubicin (dox) versus control (ctrl) treated cardiomyocytes. SLCO1A2 and SLCO1B3 indicated by blue and red arrows, respectively. (c), and (d) Enrichment scores for individual sgRNAs targeting SLCO1A2 and SLCO1B3. (e) Western-blot for protein expression of SLCO1A2 and SLCO1B3 in human iPS derived cardiomyocytes. Full-length blots are included in Supplementary Figure 10a–b. (f) Western-blot for protein expression of SLCO1A2 and SLCO1B3 in human heart tissue. Full-length blots are included in Supplementary Figure 10c–d.

Figure 4

SLCO1A2 mediates doxorubicin-toxicity in iPS derived cardiomyocytes. (a) Dose response for doxorubicin toxicity in cells expressing control Scramble shRNA, or two sequence independent shRNAs targeting SLCO1A2 in iPS derived cardiomyocytes or BT549 breast cancer cells (**: p < 0.01 vs scramble shRNA). (b) Doxorubicin-toxicity in cells treated with the SLCO1A2 inhibitor naringin in iPS derived cardiomyocytes or BT549 cancer cells (**: p < 0.01). (c) Doxorubicin toxicity (n = 8 per condition) and (d) uptake (n = 3 per condition) in cells over-expressing SLCO1A2 (**: p < 0.01). (a–d) p from student’s T-test.