Table 1.

Milestone neurological disease studies that made use of genome-scale CRISPR-Cas9 screens

Disease	Screen objective	Screen type	Methodology	Results	Reference
ALS	To find regulators of SQSTM1/p62	Activation	Lentiviral transduction of sgRNA library into human neuroglioma H4 cells expressing GFP-tagged SQSTM1 and Cas9. Selection through FACS. A mini-pool screen followed to verify top hits.	Identified the MTOR signaling pathway and the entire macroautophagy machinery as key regulators of SQSTM1. Also uncovered HNRNPM, SLC39A14, SRRD, PGK1, and the ufmylation cascade as modulators.	[107]
PD	To find transcriptional networks that protect against alpha-synuclein toxicity	Activation	Doxycycline (Dox)-inducible (Tet-ON) dCas9-VP64 expression cassette was integrated into yeast cells expressing YFP-tagged alpha-synuclein. Cells were transformed with sgRNA library and selected for survival. Validation in yeast and SHSY5Y cells.	Identified crisprTFs that were protective against alpha-synuclein toxicity that modulate protein quality control, ER/Golgi trafficking, lipid metabolism, mitochondrial function, and stress response.	[30]
PD	To elucidate the effects of cellular PARKIN abundance on downstream processes	Knockout	Lentiviral transduction of sgRNA library into human HEK-derived JumpIN TI 293 cells that express endogenous GFP-tagged PARKIN. Selection through FACS. Top hits were verified in iPSC iNGN cells.	Identified genes that regulate PARKIN gene expression positively and negatively. Specifically, transcriptional repressor THAP11 can repress PARKIN and impact pUb accumulation.	[28]
ALS	To find genetic modifiers of C9orf72 peptide repeat toxicity	Knockout	Lentiviral transduction of sgRNA library into human myelogenous leukemia K562 cells. Treatment with synthetic or lentivirally transduced DPR proteins in two separate screens. Validation in subset screen based on mouse primary neurons. Top hits from both screens were validated in mouse dorsal root ganglion axons and iPSCs from patients.	Uncovered potent candidate modifiers of DPR toxicity. Specifically, TMX2 was observed to modify DPR toxicity and exhibited promise as a therapeutic target.	[31]
Zika virus	To find host encoded proteins that mediate Zika virus infection	Knockout	Lentiviral transduction of sgRNA library into human iPSC derived neuroprogenitor cells. Zika virus infection causes majority of cells to die 48 h postinfection. Validation in a subset screen based on human iPSC and ESC cells validated top-ranked hits from initial screen.	Identified gene products with roles in heparan sulfation, ER translocation and proteostasis, Golgi-based glycosylation and the cellular response to interferon, as mediators of Zika virus-dependent cell death	[108]