Project description:Human chromosomes are pervasively transcribed, but systematic understanding of coding and lncRNA genome function in cell differentiation is lacking. Using CRISPR interference (CRISPRi) in human induced pluripotent stem cells, we performed dual genome-wide screens – assessing 18,905 protein-coding and 10,678 lncRNA loci – and identified 419 coding and 201 lncRNA genes that regulate neural induction. Integrative analyses revealed distinct properties of coding and lncRNA genome function, including a 10-fold enrichment of lncRNA genes for roles in differentiation compared to proliferation. Further, we applied Perturb-Seq to obtain granular insights into neural induction phenotypes. While most coding hits stalled or aborted differentiation, lncRNA hits were enriched for the genesis of diverse cellular states, including those outside the neural lineage. In addition to providing a rich resource (danlimlab.shinyapps.io/dualgenomewide) for understanding coding and lncRNA gene function in development, these results indicate that the lncRNA genome regulates lineage commitment in a manner fundamentally distinct from coding genes.
Project description:The human genome produces thousands of long non-coding RNAs (lncRNAs) – transcripts >200 nucleotides long that do not encode proteins. While critical roles in normal biology and disease have been revealed for a subset of lncRNAs, the function of the vast majority remains untested. Here, we developed a CRISPR interference (CRISPRi) platform targeting 16,401 lncRNA loci in 7 diverse cell lines including 6 transformed cell lines and human induced pluripotent stem cells (iPSCs). Large-scale screening identified 499 lncRNA loci required for robust cellular growth, of which 89% showed growth modifying function exclusively in one cell type. We further found that lncRNA knockdown can perturb complex transcriptional networks in a cell type-specific manner. These data underscore the functional importance and cell type-specificity of many lncRNAs.
Project description:Induced pluripotent stem cell (iPSC) derived organoid systems provide models to study human organ development. Single-cell transcriptome sequencing enables highly-resolved descriptions of cell state heterogeneity within these systems and computational methods can reconstruct developmental trajectories. However, new approaches are needed to directly measure lineage relationships in these systems. Here we establish an inducible dual channel lineage recorder, iTracer, that couples reporter barcodes, inducible CRISPR/Cas9 scarring, and single-cell transcriptomics to analyze state and lineage relationships in iPSC-derived systems. This data set include the iTracer-perturb data of one cerebral organoid with simultaneous TSC2 perturbation and lineage recording.
Project description:We tried to understand the N-glycoproteom molecular differences between induced pluripotent stem cells and embryonic stem cells. This is important to improve the reprograming process and induced pluripotency in the context of post translational protein N-glycosylation
Project description:We develop a system for bidirectional epigenetic editing (CRISPRai), in which orthogonal activating (CRISPRa) and repressive (CRISPRi) perturbations are applied simultaneously to multiple loci the same cell. We perform Perturb-seq, a method for single-cell RNA-seq paired with CRISPR guide RNA (gRNA) detection, on cells perturbed using CRISPRai for a panel of pairwise combinations of genes in K562 cells.
Project description:Genome-wide analysis of chromatin state in extended pluripotent stem cells, primed pluripotent stem cells, and naïve pluripotent stem cells
Project description:Perturb-seq is a powerful approach to systematically assess how genes and enhancers impact the molecular and cellular pathways of development and disease. However, technical challenges have limited its application in stem cell-based systems. Here, we benchmarked Perturb-seq across multiple CRISPRi modalities, on diverse genomic targets, in multiple human pluripotent stem cells, during directed differentiation to multiple lineages, and across multiple sgRNA delivery systems. To ensure cost-effective production of large-scale Perturb-seq datasets as part of the Impact of Genomic Variants on Function (IGVF) consortium, our optimized protocol dynamically assesses experiment quality across the weeks-long procedure. Our analysis of 1,996,260 sequenced cells across benchmarking datasets reveals shared regulatory networks linking disease-associated enhancers and genes with downstream targets during cardiomyocyte differentiation. This study establishes open tools and resources for interrogating genome function during stem cell differentiation.
