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:we performed lentiviral CRISPR interference (CRISPRi) by recruiting dCas9 fused with the KRAB domain to the CSMD1 enhancer (fam3) in the neuronal precursor cell line – Lund human mesencephalic (LUHMES). Given that the expression of CSMD1 was not detectable in LUHMES cells we differentiated these cells into neurons. Differentiated neurons with CRISPRi of CSMD1 enhancer showed significantly higher expression of CSMD1 than control.
Project description:We performed single cell transcriptomic profiling of induced human pluripotent stem cells (iPSCs)-derived type 2 alveolar epithelial cells (iAT2). iPSCs stably expressed CRISPRi (dCas9-KRAB) under the control of doxycyline. iAT2s were transduced with a lentivirus expressing gRNA targeting the transcriptional start site of DSP. Cells were treated with or without doxycyline to intiate CRISPRi-knockdown. Prior to capture, cells were labelled with hashing antibodies (HTO). Cells were captured for 10x Genomics Single cell capture (V3 protocol), and GEX and HTO libraries were sequenced. HTODemux function was used to demultiplex the samples. Knockdown of DSP caused cells to cluster separately from wild-type cells.
Project description:We performed bulk transcriptomic profiling of induced human pluripotent stem cells (iPSCs)-derived type 2 alveolar epithelial cells (iAT2). iPSCs stably expressed CRISPRi (dCas9-KRAB) under the control of doxycyline. iAT2s were transduced with a lentivirus expressing gRNA targeting the transcriptional start site of ADGRG6. Cells were treated with or without doxycyline to intiate CRISPRi-knockdown. Cells were plated at an air-liquid interface, then subsequently exposed to air or 5% cigarette smoke using a VitroCell smoke robot. Cells were harvested for bulk RNA sequencing 8 hours post cigarette smoke exposure
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:We performed single cell transcriptomic profiling of induced human pluripotent stem cells (iPSCs)-derived type 2 alveolar epithelial cells (iAT2). iPSCs stably expressed CRISPRi (dCas9-KRAB) under the control of doxycyline. iAT2s were transduced with a lentivirus expressing gRNA targeting the transcriptional start site of ADGRG6. Cells were treated with or without doxycyline to intiate CRISPRi-knockdown. Prior to capture, cells were labelled with hashing antibodies (HTO). Cells were captured for 10x Genomics Single cell capture (V3 protocol), and GEX and HTO libraries were sequenced. HTODemux function was used to demultiplex the samples. Knockdown of ADGRG6 caused cells to cluster separately from wild-type cells.
Project description:RNA-seq was performed to identify genes affected by CRISPRi (dCas9-KRAB) with sgRNA (sgSFTPB#2) targeting the first intronic region of human SFTPB bound by NKX2-1 in an unbiased genome-wide fashion using A549 lung carcinoma cells that constitutively express dCas9-KRAB and NKX2-1.
Project description:RNA-seq was performed to identify genes affected by CRISPRi (dCas9-KRAB) with sgRNA (sgTGFB1_B9D2) targeting a region that contains rs1800469 in an unbiased genome-wide fashion using H441 lung papillary adenocarcinoma cells.