CRISPR/Cas9 has revolutionized our ability to engineer genomes and conduct genome-wide screens in human cells. Whereas some cell types are amenable to genome engineering, genomes of human pluripotent stem cells (hPSCs) have been difficult to engineer, with reduced efficiencies relative to tumour cell lines or mouse embryonic stem cells. Here, using hPSC lines with stable integration of Cas9 or transient delivery of Cas9-ribonucleoproteins (RNPs), we achieved an average insertion or deletion (indel) efficiency greater than 80%. This high efficiency of indel generation revealed that double-strand breaks (DSBs) induced by Cas9 are toxic and kill most hPSCs. In previous studies, the toxicity of Cas9 in hPSCs was less apparent because of low transfection efficiency and subsequently low DSB induction. The toxic response to DSBs was P53/TP53-dependent, such that the efficiency of precise genome engineering in hPSCs with a wild-type P53 gene was severely reduced. Our results indicate that Cas9 toxicity creates an obstacle to the high-throughput use of CRISPR/Cas9 for genome engineering and screening in hPSCs. Moreover, as hPSCs can acquire P53 mutations, cell replacement therapies using CRISPR/Cas9-enginereed hPSCs should proceed with caution, and such engineered hPSCs should be monitored for P53 function.

Zika virus (ZIKV) can cross the placental barrier, resulting in infection of the fetal brain and neurological defects including microcephaly. The cellular tropism of ZIKV and the identity of attachment factors used by the virus to gain access to key cell types involved in pathogenesis are under intense investigation. Initial studies suggested that ZIKV preferentially targets neural progenitor cells (NPCs), providing an explanation for the developmental phenotypes observed in some pregnancies. The AXL protein has been nominated as a key attachment factor for ZIKV in several cell types including NPCs. However, here we show that genetic ablation of AXL has no effect on ZIKV entry or ZIKV-mediated cell death in human induced pluripotent stem cell (iPSC)-derived NPCs or cerebral organoids. These findings call into question the utility of AXL inhibitors for preventing birth defects after infection and suggest that further studies of viral attachment factors in NPCs are needed.

Here we report Digital RNA with pertUrbation of Genes (DRUG-seq), a high-throughput platform for drug discovery. Pharmaceutical discovery relies on high-throughput screening, yet current platforms have limited readouts. RNA-seq is a powerful tool to investigate drug effects using transcriptome changes as a proxy, yet standard library construction is costly. DRUG-seq captures transcriptional changes detected in standard RNA-seq at 1/100th the cost. In proof-of-concept experiments profiling 433 compounds across 8 doses, transcription profiles generated from DRUG-seq successfully grouped compounds into functional clusters by mechanism of actions (MoAs) based on their intended targets. Perturbation differences reflected in transcriptome changes were detected for compounds engaging the same target, demonstrating the value of using DRUG-seq for understanding on and off-target activities. We demonstrate DRUG-seq captures common mechanisms, as well as differences between compound treatment and CRISPR on the same target. DRUG-seq provides a powerful tool for comprehensive transcriptome readout in a high-throughput screening environment.

Steroid hormones act, through their respective nuclear receptors, to regulate target gene expression. Despite their critical role in development, physiology, and disease, however, it is still unclear how these systemic cues are refined into tissue-specific responses. We identified a mutation in the evolutionarily conserved DEAD box RNA helicase belle/DDX3 that disrupts a subset of responses to the steroid hormone ecdysone during Drosophila melanogaster metamorphosis. We demonstrate that belle directly regulates translation of E74A, an ets transcription factor and critical component of the ecdysone-induced transcriptional cascade. Although E74A mRNA accumulates to abnormally high levels in belle mutant tissues, no E74A protein is detectable, resulting in misregulation of E74A-dependent ecdysone response genes. The accumulation of E74A mRNA in belle mutant salivary glands is a result of auto-regulation, fulfilling a prediction made by Ashburner nearly 40 years ago. In this model, Ashburner postulates that, in addition to regulating secondary response genes, protein products of primary response genes like E74A also inhibit their own ecdysone-induced transcription. Moreover, although ecdysone-triggered transcription of E74A appears to be ubiquitous during metamorphosis, belle-dependent translation of E74A mRNA is spatially restricted. These results demonstrate that translational control plays a critical, and previously unknown, role in refining transcriptional responses to the steroid hormone ecdysone.

Highlights d A universal and scalable genetic platform in hPSCs for general use across all lineages d Robust knockout efficiencies translate into high-performance screening at genome scale d Stem cell-specific components of TP53 and OCT4 genetic networks in hPSCs are identified d Validation of PMAIP1 and PAWR function in sensitivity to DNA damage or dissociation