Patient Brain Organoids Identify a Link between the 16p11.2 Copy Number Variant and the Gene.

Copy number variants (CNVs) that delete or duplicate 30 genes within the 16p11.2 genomic region give rise to a range of neurodevelopmental phenotypes with high penetrance in humans. Despite the identification of this small region, the mechanisms by which 16p11.

Natural variation in gene expression and viral susceptibility revealed by neural progenitor cell villages.

Human genome variation contributes to diversity in neurodevelopmental outcomes and vulnerabilities; recognizing the underlying molecular and cellular mechanisms will require scalable approaches. Here, we describe a "cell village" experimental platform we used to analyze genetic, molecular, and phenotypic heterogeneity across neural progenitor cells from 44 human donors cultured in a shared in vitro environment using algorithms (Dropulation and Census-seq) to assign cells and phenotypes to individual donors. Through rapid induction of human stem cell-derived neural progenitor cells, measurements of natural genetic variation, and CRISPR-Cas9 genetic perturbations, we identified a common variant that regulates antiviral IFITM3 expression and explains most inter-individual variation in susceptibility to the Zika virus.

DRUG-seq Provides Unbiased Biological Activity Readouts for Neuroscience Drug Discovery.

Unbiased transcriptomic RNA-seq data has provided deep insights into biological processes. However, its impact in drug discovery has been narrow given high costs and low throughput. Proof-of-concept studies with Digital RNA with pertUrbation of Genes (DRUG)-seq demonstrated the potential to address this gap.

Genome-Scale CRISPR Screens Identify Human Pluripotency-Specific Genes.

Human pluripotent stem cells (hPSCs) generate a variety of disease-relevant cells that can be used to improve the translation of preclinical research. Despite the potential of hPSCs, their use for genetic screening has been limited by technical challenges. We developed a scalable and renewable Cas9 and sgRNA-hPSC library in which loss-of-function mutations can be induced at will.

p53 inhibits CRISPR-Cas9 engineering in human pluripotent stem cells.

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%.

Combining NGN2 Programming with Developmental Patterning Generates Human Excitatory Neurons with NMDAR-Mediated Synaptic Transmission.

Transcription factor programming of pluripotent stem cells (PSCs) has emerged as an approach to generate human neurons for disease modeling. However, programming schemes produce a variety of cell types, and those neurons that are made often retain an immature phenotype, which limits their utility in modeling neuronal processes, including synaptic transmission. We report that combining NGN2 programming with SMAD and WNT inhibition generates human patterned induced neurons (hpiNs).

Genetic Ablation of AXL Does Not Protect Human Neural Progenitor Cells and Cerebral Organoids from Zika Virus Infection.

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.

A deleterious Nav1.1 mutation selectively impairs telencephalic inhibitory neurons derived from Dravet Syndrome patients.

Dravet Syndrome is an intractable form of childhood epilepsy associated with deleterious mutations in SCN1A, the gene encoding neuronal sodium channel Nav1.1. Earlier studies using human induced pluripotent stem cells (iPSCs) have produced mixed results regarding the importance of Nav1.

The let-7/LIN-41 pathway regulates reprogramming to human induced pluripotent stem cells by controlling expression of prodifferentiation genes.

Reprogramming differentiated cells into induced pluripotent stem cells (iPSCs) promotes a broad array of cellular changes. Here we show that the let-7 family of microRNAs acts as an inhibitory influence on the reprogramming process through a regulatory pathway involving prodifferentiation factors, including EGR1. Inhibiting let-7 in human cells promotes reprogramming to a comparable extent to c-MYC when combined with OCT4, SOX2, and KLF4, and persistence of let-7 inhibits reprogramming.

The zinc finger protein Zn72D and DEAD box helicase Belle interact and control maleless mRNA and protein levels.

Background: The Male Specific Lethal (MSL) complex is enriched on the single X chromosome in male Drosophila cells and functions to upregulate X-linked gene expression and equalize X-linked gene dosage with XX females. The zinc finger protein Zn72D is required for productive splicing of the maleless (mle) transcript, which encodes an essential subunit of the MSL complex. In the absence of Zn72D, MLE levels are decreased, and as a result, the MSL complex no longer localizes to the X chromosome and dosage compensation is disrupted.

Zinc finger protein Zn72D promotes productive splicing of the maleless transcript.

In organisms with sex chromosomes, dosage compensation equalizes gene expression between the sexes. In Drosophila melanogaster males, the male-specific lethal (MSL) complex of proteins and two noncoding roX RNAs coat the X chromosome, resulting in a twofold transcriptional upregulation to equalize gene expression with that of females. How MSL complex enrichment on the X chromosome is regulated is not well understood.

Developmental regulation of Suz 12 localization.

Chromatin modifications are among the epigenetic alterations essential for genetic reprogramming during development. The Polycomb group (PcG) gene family mediates chromatin modifications that contribute to developmentally regulated transcriptional silencing. Trimethylation of histone H3 on lysine 27, mediated by a PcG protein complex consisting of Eed, Ezh2, and Suz12, is integral in differentiation, stem cell self-renewal, and tumorigenesis.

Chromatin modifications on the inactive X chromosome.

In female mammals, one X chromosome is transcriptionally silenced to achieve dosage compensation between XX females and XY males. This process, known as X-inactivation, occurs early in development, such that one X chromosome is silenced in every cell. Once X-inactivation has occurred, the inactive X chromosome is marked by a unique set of epigenetic features that distinguishes it from the active X chromosome and autosomes.

Role of histone H3 lysine 27 methylation in X inactivation.

The Polycomb group (PcG) protein Eed is implicated in regulation of imprinted X-chromosome inactivation in extraembryonic cells but not of random X inactivation in embryonic cells. The Drosophila homolog of the Eed-Ezh2 PcG protein complex achieves gene silencing through methylation of histone H3 on lysine 27 (H3-K27), which suggests a role for H3-K27 methylation in imprinted X inactivation. Here we demonstrate that transient recruitment of the Eed-Ezh2 complex to the inactive X chromosome (Xi) occurs during initiation of X inactivation in both extraembryonic and embryonic cells and is accompanied by H3-K27 methylation.