Project description:All bulk CRISPR based screens CD2 and B2M CRISPRi tiling screens (primary human CD8 T cells), IL2RA CRISPRa tiling screens (Jurkats), CRISPRi/a TF screens (primary human CD8 T cells), and CRISPR TFome KO (primary human T cells)
Project description:We demonstrate that vector designs for such screens that rely on cis linkage of guides and distally located barcodes suffer from swapping of intended guide-barcode associations at rates approaching 50% due to template switching during lentivirus production, greatly reducing sensitivity.
Project description:This is data for the evaluation of a new way of counting sgRNAs in CRISPR screens using padlock probes and UMIs. It is compared to the typical PCR-based approach. In particular, a dropout screen was performed in MiaPaCa-2 cells using the Human Kinome CRISPR pooled library (Addgene #75314)
Project description:CRISPR/Cas9-based functional genomics have transformed our ability to elucidate mammalian cell biology. Most previous CRISPR-based screens were implemented in cancer cell lines, rather than healthy, differentiated cells. Here, we describe a CRISPR interference (CRISPRi)-based platform for genetic screens in human neurons derived from induced pluripotent stem cells (iPSCs). We demonstrate robust and durable knockdown of endogenous genes in such neurons, and present results from three complementary genetic screens. A survival-based screen revealed neuron-specific essential genes and a small number of genes that improved neuronal survival upon knockdown. A screen with a single-cell transcriptomic readout uncovered several examples for genes knockdown of which had dramatically different cell-type specific consequences. A longitudinal imaging screen detected distinct consequences of gene knockdown on neuronal morphology. Our results highlight the potential of interrogating cell biology in iPSC-derived differentiated cell types and provide a platform for the systematic dissection of normal and disease states of neurons.
Project description:Uncovering the genetic basis of molecular processes is essential for understanding a broad range of biological phenomena. CRIPSR-Cas9 approaches enable this discovery through programable and systematic profiling of regulatory genes underlying diverse cellular behaviors. A key challenge for dissecting genetic networks is expanding CRISPR-based screens to interrogate specific phenotypes with high precision. Here, we use a quantitative, sequencing-based CRISPRi platform to enhance the scope and sensitivity of genome-wide screens. We first systematically explore how technical variations distort guide effects and correct for these factors by using RNA reporters and normalizers expressed from closely matched promoters. We then combine this platform with a recombinase-based integration system to interrogate protein and RNA-level phenotypes. We find our approach accurately captures known regulators of protein or RNA quality control with high accuracy and minimal background. These barcode-based CRISPR systems provide a powerful and generalizable platform for dissecting critical cellular regulatory pathways.