Genome-wide CRISPRi/a screens in human neurons link lysosomal failure to ferroptosis

Abstract

SUMMARYSingle-cell transcriptomics provide a systematic map of gene expression in different human cell types. The next challenge is to systematically understand cell-type specific gene function. The integration of CRISPR-based functional genomics and stem cell technology enables the scalable interrogation of gene function in differentiated human cells. Here, we present the first genome-wide CRISPR interference and CRISPR activation screens in human neurons.We uncover pathways controlling neuronal response to chronic oxidative stress, which is implicated in neurodegenerative diseases. Unexpectedly, knockdown of the lysosomal protein prosaposin strongly sensitizes neurons, but not other cell types, to oxidative stress by triggering the formation of lipofuscin, a hallmark of aging, which traps iron, generating reactive oxygen species and triggering ferroptosis. We also determine transcriptomic changes in neurons following perturbation of genes linked to neurodegenerative diseases. To enable the systematic comparison of gene function across different human cell types, we establish a data commons named CRISPRbrain.

Mind the translational gap: using iPS cell models to bridge from genetic discoveries to perturbed pathways and therapeutic targets

“…MPRAs Saturation mutagenesis with MPRAs Mutagenesis on disease-associated gene promoters and enhancers Kircher et al [175] Targeted variants mutagenesis with MPRAs Functional dissection of common genetic Variation Ulirsch et al [176] CRISPR screens Perturb-seq CRISPR screen combined with single cell RNA-seq (scRNA-seq) Dixit et al [122] CRISPRa/i screens CRISPR screens modulating gene expression Tian et al [177] CREST-seq Cis-regulatory elements scan by tiling-deletions Diao et al [178] MOSAIC-seq Genome-wide CRISP/i screens targeting enhancers Xie et al [179] CRISPR-flowFISH RNA-FISH coupled to genome-wide CRISP/i screens targeting enhancers Fulco et al [180] CRISPR targeting CRISPRa/i gene targeting CRISPRa/i-mediated modulation of selected regulatory regions Gasperini et al [181] CRISPR-mediated allelic replacement CRISPR-mediated nonhomologous end joining (NHEJ) or homology-directed repair (HDR)…”

Functional annotation of lncRNA in high-throughput screening

“…The cellular phenotypes assessed after genetic perturbation are diverse ( Figure 1 C), including survival advantage for robust cell growth [ 15 , 17 , 25 ], after drug treatments [ 18–20 ] or with fractionated radiation [ 21 ]. By combining with cell sorting, a wide variety of phenotypes can be measured, such as pluripotency maintenance [ 15 , 23 , 72 ], differentiation [ 73 ], protein transport [ 74 ], oxidative stress [ 75 ], and many more.…”

“…Formaldehyde fixation can reduce the degree of cell loss while de-crosslinking is required to rescue the genomic DNA for PCR library construction [ 15 ]. Indeed, many screens rely on expression of exogenous genes carrying fluorescent signals [ 73 , 74 ] or fluorescent probe live trackers [ 75 ]. For example, Liu and colleagues [ 73 ] performed a CRISPRa screen in mouse ES cells, where the cell surface marker hCD8 was inserted downstream of Tubb3.…”

Editing the Epigenome in Neurodegenerative Diseases

“…Researchers created a database called CRISPRbrain (https://crisprbrain.org/) combining the results of genetic screenings for various phenotypes in different types of human cells, conducted by different research groups. This is an extremely valuable resource for comparing phenotype data affecting neurodegeneration [44].…”