Genome-wide CRISPRi/a screens in human neurons link lipid imbalance to ferroptosis

Neurons are unique among all human cell types in their high energy demand, long lifespans and rich lipid contents. As such, neurons exhibit unique vulnerability to oxidative stress caused by redox imbalance in aging and neurodegenerative diseases (NDDs). To systematically identify regulators of neuronal survival under oxidative stress and regulators of neuronal redox homeostasis, we conducted multiple survival- and FACS-based genome-wide screens in human iPSC-derived neurons, using our functional genomics toolkit including a previously established CRISPRi approach and a newly developed CRISPRa approach. So far, these are the first genome-wide screens in human neurons. Our results revealed that inhibiting glycosphingolipids (GSLs) degradation by depletion of prosaposin (PSAP) drives the formation of lipofuscins in neurons which leads to iron accumulation and strongly induces ROS production that oxidizing lipids and leads to neuronal ferroptosis under oxidative stress. We also conducted single cell CROP-seq screens that revealed transcriptomic signatures of NDD-associated genes. These datasets are freely available through our open-access database CRISPRbrain. Overall design: For the CROP-seq experiments, we included 184 genes for CRISPRi and 100 genes for CRISPRa, which were hits in at least one of our genome-wide pooled screens and were also associated with human neurodegenerative diseases (NDDs). We curated a list of NDD-associated genes based on the literature and the DisGeNET database (https://www.disgenet.org/). The CROP-seq libraries included 2 sgRNAs per gene plus 6 non-targeting control sgRNAs, for a total of 374 sgRNAs for CRISPRi and 206 sgRNAs for CRISPRa. Top and bottom strands of sgRNA oligos were synthesized (Integrated DNA Technologies) and annealed in an arrayed format. The annealed sgRNAs were then pooled in equal amounts and ligated into our optimized CROP-seq vector (Gilbert et al., 2014).The CROP-seq experiments were carried out similarly as previously described (Tian et al., 2019). Briefly, Day 0 CRISPRi and CRISPRa neurons were infected by the corresponding CROP-seq sgRNA library at a MOI of 0.1-0.2, followed by puromycin selection at 4 µg/ml for 3 days and recovery. On Day 10, neurons were dissociated with Papain and approximately 98,000 CRISPRi neurons and 50,000 CRISPRa neurons were loaded into 10X chips with about 25,000 input cells per lane. Sample preparations were performed using the ChromiumNext GEM Single Cell 3' Reagent Kits v3.1 (10X Genomics; Cat. No.PN-1000121) according to the manufacturer's protocol. To facilitate sgRNA assignment, sgRNA-containing transcripts were additionally amplified by hemi-nested PCR reactions as described (Tian et al., 2019). The sgRNA-enrichment libraries were separately indexed and sequenced as spike-ins alongside the whole- transcriptome scRNA-seq libraries using a NovaSeq 6000 using the following configuration: Read 1: 28, i7 index: 8, i5 index: 0, Read 2: 91. 3'-tag RNA-seq was performed to obtain gene expression profiles of WT and PSAP KO neurons cultured in the medium lack of antioxidants. Each genotype has 3 replicates. Raw sequencing reads from 3'-tag RNA-seq were mapped to the human reference transcriptome (GRCh38, Ensembl Release 97) using Salmon (v.0.14.139) with the '–noLengthCorrection' option to obtain transcript abundance counts. Gene-level count estimates were obtained using tximport (v.1.8.040) with default settings. Subsequently, genes with more than 10 counts were retained for differential gene-expression analysis, and adjusted P values (Padj) were calculated using DESeq2 (v.1.20.041).