Project description:~1500 K562 cells expressing dCas9-KRAB were profiled on the 10x Genomics 3' v3 droplet-based scRNA-seq platform. The resulting library was sequenced on the Illumina and Ultima sequencing platforms.
Project description:To compare chromatin accessibility across three primate species, between wild-type (WT) and genetically modified induced pluripotent stem cell (iPSC) lines, and between the iPSC state and neural precursor cells (NPCs) derived from these iPSCs, we generated ATAC-seq data from nine primate samples. The samples included two gorilla WT iPSC samples and one gorilla KRAB-dCas9 iPSC sample (all from the same individual), one orangutan WT iPSC sample, one orangutan KRAB-dCas9 iPSC sample and two orangutan NPC samples (from two different individuals), and one cynomolgus macaque WT iPSC sample and one cynomolgus macaque KRAB-dCas9 iPSC sample (from the same individual). The gorilla and orangutan iPSCs were derived from urinary stem cells (Geuder et al. 2021), while the cynomolgus macaque iPSCs were derived from skin-fibroblasts. The KRAB-dCas9 iPS cell lines were created by stably integrating dox-inducible KRAB-dCas9-HA-P2A-mCherry construct at the AAVS1 locus (Edenhofer et al. 2024). NPCs were obtained by the directed differentiation of iPSCs via dual-SMAD inhibition (Chambers et al. 2009; Ohnuki et al. 2014). ATAC-seq libraries were generated using the Omni-ATAC protocol (Corces et al. 2017) with minor modifications.
Project description:Bulk RNA sequencing was performed to assess transcriptome-wide changes associated with dCas9-KRAB mediated CRISPR interference (CRISPRi) of telomerase reverse transcriptase (TERT) in H1299 non-small cell lung cancer cells. H1299 cells were transduced with a lentiviral vector co-expressing dCas9-KRAB and either no guide RNA (non-targeting control) or a TERT-promoter-targeting single-guide RNA, selected with puromycin, and harvested at day 33 after transduction. Differential expression analysis was used to evaluate the specificity of sustained TERT repression.
Project description:Comparisons of molecular phenotypes across primates provide unique information to understand human biology and evolution and single-cell RNA-seq CRISPR interference screens are a powerful approach to analyze them. Here, we generate and validate three human, three gorilla and two cynomolgus iPS cell lines that carry a dox-inducible KRAB-dCas9 construct in the AAVS1 locus. We show that despite variable expression levels of KRAB-dCas9 among lines, comparable downregulation of target genes and comparable phenotypic effects are observed in a single-cell RNA-seq CRISPR interference screen. Hence, we provide valuable resources for performing and further extending CRISPRi screens in human and non-human primates.
Project description:Comparisons of molecular phenotypes across primates provide unique information to understand human biology and evolution and single-cell RNA-seq CRISPR interference screens are a powerful approach to analyze them. Here, we generate and validate three human, three gorilla and two cynomolgus iPS cell lines that carry a dox-inducible KRAB-dCas9 construct in the AAVS1 locus. We show that despite variable expression levels of KRAB-dCas9 among lines, comparable downregulation of target genes and comparable phenotypic effects are observed in a single-cell RNA-seq CRISPR interference screen. Hence, we provide valuable resources for performing and further extending CRISPRi screens in human and non-human primates.
Project description:Infinium MethylationEPIC (850K) BeadChip data for wildtype SUM159 cells (2 replicates), SUM159 cells transfected with plV-KRAB (dCas9 CRISPRi system; 3 replicates), and SUM159 cells transfected with dCas9-KRAB and 4 targeting gRNAs against the ZEB1 promoter (3 replicates)
Project description:Despite the revolutionary impact of genome engineering tools in medicine, the safe and effective intracellular delivery of CRISPR remains a major obstacle for clinical applications. Here, we utilize precision molecular targeting and delivery strategies based on CRISPR/dCas9 systems adapted for epigenetic repression (dCas9-KRAB) to silence oncogenic drivers with high selectivity. As proof-of-principle, we target the EWSR1-FLI1 translocation, which encodes a chimeric and hard-to-drug oncogenic transcription factor driving approximately 85% of the cases of Ewing Sarcoma (EWS)-an aggressive childhood malignancy. We describe the development of a programmable non-viral polymeric system for the delivery of dCas9-KRAB as ribonucleoprotein (RNP) payloads for selective EWSR1-FLI1 repression. We demonstrate highly efficient intracellular delivery of RNPs loaded in polyamide-amine (PAMAM) polymers functionalized by guanidino groups, resulting in robust silencing of EWSR1-FLI1 both in established cell line xenografts and in EWS-related patient-derived xenografts (PDXs) of EWS. We show that silencing of EWSR1–FLI1 is accompanied by potent anti-tumor effects. To our knowledge, this represents the first effective non-viral platform for in vivo delivery of dCas9-KRAB/RNPs, which could be adapted for the repression of any oncogene. We further outline dCas9/RNP formulations for future therapeutic applications to treat poor-prognosis cancers driven by hard-to-drug oncogenes.