Pitfalls in Single Clone CRISPR-Cas9 Mutagenesis to Fine-map Regulatory Intervals
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ABSTRACT: We report gene expression of four and three single-cell derived clones from HL60 and HL60/S4 cell lines, respectively, and the gene expression of bulk parental lines. We also report gene expression of control and mnocyte-derived HL60 (n=2, n=2, respectively) and HL60/S4 (n=2, n=2, respectively) single-cell clones, and control and neutrophil-derived HL60 (n=4, n=4, respectively) and HL60/S4 (n=4, n=4, respectively) single-cell clones.
Project description:The human promyelocytic cell line HL60/S4 can be differentiated into a granulocytic form with the addition of all-trans retinoic acid for 4 days. We investigated the conserved and differentiated responses to TNF in the granulocytic and promyelocytic forms of HL60/S4 cells.
Project description:The majority of genetic variants affecting complex traits map to regulatory regions of genes, and typically lie in credible intervals of 100 or more SNPs. Fine mapping of the causal variant(s) at a locus depends on assays that are able to discriminate the effects of polymorphisms or mutations on gene expression. Here, we evaluated a moderate-throughput CRISPR-Cas9 mutagenesis approach, based on replicated measurement of transcript abundance in single-cell clones, by deleting candidate regulatory SNPs, affecting four genes known to be affected by large-effect expression Quantitative Trait Loci (eQTL) in leukocytes, and using Fluidigm qRT-PCR to monitor gene expression in HL60 pro-myeloid human cells. We concluded that there were multiple constraints that rendered the approach generally infeasible for fine mapping. These included the non-targetability of many regulatory SNPs, clonal variability of single-cell derivatives, and expense. Power calculations based on the measured variance attributable to major sources of experimental error indicated that typical eQTL explaining 10% of the variation in expression of a gene would usually require at least eight biological replicates of each clone. Scanning across credible intervals with this approach is not recommended.
Project description:Precise identification of causal variants within credible intervals of eQTL associations is needed to identify regulatory GWAS variants. We show that CROPseq, namely multiplex CRISPR-Cas9 genome editing combined with single cell RNAseq, is a viable strategy for fine mapping regulatory SNPs. Mutations were induced nearby 67 SNPs in three genes, two of which, rs2251039 and rs17523802, significantly altered CISD1 and PARK7 expression, respectively, and overlap with chromatin accessibility peaks.
Project description:RNA-seq of embryonic mammary progenitor cell lines. Embryonic mammary progenitor cell (eMPC) clones were derived from E12.5-stage mammary organs. After microdissection, mammary primordial 3 was cultured on thick basement membrane extract for 4 weeks. After enzymatic dissociation eMPC swere plated and expanded in 2D culture to obtain a pool of eMPCs. eMPC cells were subject to single cell sorting using FACS. Clones were expanded as single-cell derived clones (eMPC1, 2, etc.) to create the eighteen single cell-derived clones described in this study.
Project description:HSC-2 (hepatic stellate cells line from rat) were stably transfected with rno-miR-146a. Three different clones were selected (S1, S4, S5). We used Affymetrix rat genome RAT230 2.0 chip to monitor global transcriptome changes.