Protein-encoding nucleotide repeat expansion diseases, including polyglutamine (polyQ) and polyglycine (polyG) diseases, are characterized by the accumulation of aggregation-prone proteins. In the polyQ diseases, including Huntington’s disease and several spinocerebellar ataxias, substantial prior evidence supports a pathogenic role for mutant polyQ-expanded protein misfolding and aggregation, with molecular chaperones showing promise in suppressing disease phenotypes in cellular and animal models. The goal of this study is to establish a scalable cell-based model to systematically evaluate genetic modifiers of protein aggregation in both polyQ and polyG diseases. We developed FRET-based reporter systems that model polyQ and polyG aggregation in human cells and used them to perform high-throughput CRISPR interference screens targeting all known molecular chaperones. In the polyQ model, the screen identified multiple Hsp70 chaperones (HSPA8, HSPA4) and Hsp40 co-chaperones (DNAJB6, DNAJB1) previously implicated in polyQ aggregation and additionally revealed the Hsp40 co-chaperone DNAJC7 as a potent and previously unrecognized suppressor of polyQ aggregation. In contrast, in a FRET-based polyG aggregation model of neuronal intranuclear inclusion disease, CRISPRi screening showed minimal overlap of chaperone modifiers of the polyQ screen. Direct knockdown of DNAJC7 also did not affect polyG aggregation, yet overexpressed DNAJC7 co-localized with both polyQ and polyG aggregates in cells and reduced their aggregation. In addition to establishing new inducible, scalable cellular models for polyQ and polyG aggregation, this work expands the role of DNAJC7 in regulating folding of disease-associated proteins.

HEK293T cells containing CRISPRi machinery¹ were transduced with lentiviral constructs expressing doxycycline-inducible, nuclear-localized polyQ or polyG proteins that are aggregation-prone. The proteins were also fluorescently tagged (mScarlet and mNeonGreen). This fluorescence pair allows FRET-based readout of protein aggregation. We subsequently generated a monoclonal cell line for the FRET-paired polyQ or polyG inducible protein expression, and designated it as NLS-FRET-Q79 or NLS-FRET-G100, respectively. 

The cell lines were transduced with a sgRNA library consisting of 2071 sgRNAs targeting nearly all known molecular chaperones (356 genes) along with non-targeting controls, as previously described². On Day 2, with ∼30 % of the cells showing BFP positivity, the cells were passaged and replated with 2 μg/ml puromycin (Gibco, A1113803). This was repeated on day 5. On day 7, with > 70 % of cells showing BFP positivity, the cells were passaged and 20 million cells were plated without puromycin and with the addition of 2 ng/ml doxycycline. The cells were regularly passaged until day 5. The cells were dissociated with trypsin, resuspended in complete DMEM, and sorted using the BD FACSAria Fusion Cell Sorter into FRET-low (∼6 million cells) and FRET-high (∼2 million cells). Sorting and gDNA isolation for the polyQ chaperone screen was performed twice (two separate times from the same starting population of library-transduced cells). Sorting for the polyG chaperone screen was performed once. Genomic DNA was isolated using a Monarch gDNA extraction kit according to manufacturer protocols. sgRNA-encoding regions were then amplified, followed by sequencing of the protospacers by Illumina NextSeq2000.

1. Guo X, Aviles G, Liu Y, Tian R, Unger BA, Lin YT, Wiita AP, Xu K, Correia MA, Kampmann M. Mitochondrial stress is relayed to the cytosol by an OMA1-DELE1-HRI pathway. Nature. 2020 Mar;579(7799):427-432
2. Biebl MM, Delhommel F, Faust O, Zak KM, Agam G, Guo X, Mühlhofer M, Dahiya V, Hillebrand D, Popowicz GM, Kampmann M, Lamb DC, Rosenzweig R, Sattler M, Buchner J. NudC guides client transfer between the Hsp40/70 and Hsp90 chaperone systems. Mol Cell. 2022 Feb 3;82(3):555-569.e7