Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors.

CRISPR interference (CRISPRi) enables programmable, reversible, and titratable repression of gene expression (knockdown) in mammalian cells. Initial CRISPRi-mediated genetic screens have showcased the potential to address basic questions in cell biology, genetics, and biotechnology, but wider deployment of CRISPRi screening has been constrained by the large size of single guide RNA (sgRNA) libraries and challenges in generating cell models with consistent CRISPRi-mediated knockdown. Here, we present next-generation CRISPRi sgRNA libraries and effector expression constructs that enable strong and consistent knockdown across mammalian cell models. First, we combine empirical sgRNA selection with a dual-sgRNA library design to generate an ultra-compact (1-3 elements per gene), highly active CRISPRi sgRNA library. Next, we compare CRISPRi effectors to show that the recently published Zim3-dCas9 provides an excellent balance between strong on-target knockdown and minimal non-specific effects on cell growth or the transcriptome. Finally, we engineer a suite of cell lines with stable expression of Zim3-dCas9 and robust on-target knockdown. Our results and publicly available reagents establish best practices for CRISPRi genetic screening.

Ras-mutant cancers are sensitive to small molecule inhibition of V-type ATPases in mice.

Mutations in Ras family proteins are implicated in 33% of human cancers, but direct pharmacological inhibition of Ras mutants remains challenging. As an alternative to direct inhibition, we screened for sensitivities in Ras-mutant cells and discovered 249C as a Ras-mutant selective cytotoxic agent with nanomolar potency against a spectrum of Ras-mutant cancers. 249C binds to vacuolar (V)-ATPase with nanomolar affinity and inhibits its activity, preventing lysosomal acidification and inhibiting autophagy and macropinocytosis pathways that several Ras-driven cancers rely on for survival. Unexpectedly, potency of 249C varies with the identity of the Ras driver mutation, with the highest potency for KRASG13D and G12V both in vitro and in vivo, highlighting a mutant-specific dependence on macropinocytosis and lysosomal pH. Indeed, 249C potently inhibits tumor growth without adverse side effects in mouse xenografts of KRAS-driven lung and colon cancers. A comparison of isogenic SW48 xenografts with different KRAS mutations confirmed that KRASG13D/+ (followed by G12V/+) mutations are especially sensitive to 249C treatment. These data establish proof-of-concept for targeting V-ATPase in cancers driven by specific KRAS mutations such as KRASG13D and G12V.

Endoplasmic reticulum tubules limit the size of misfolded protein condensates.

The endoplasmic reticulum (ER) is composed of sheets and tubules. Here we report that the COPII coat subunit, SEC24C, works with the long form of the tubular ER-phagy receptor, RTN3, to target dominant-interfering mutant proinsulin Akita puncta to lysosomes. When the delivery of Akita puncta to lysosomes was disrupted, large puncta accumulated in the ER. Unexpectedly, photobleach analysis indicated that Akita puncta behaved as condensates and not aggregates, as previously suggested. Akita puncta enlarged when either RTN3 or SEC24C were depleted, or when ER sheets were proliferated by either knocking out Lunapark or overexpressing CLIMP63. Other ER-phagy substrates that are segregated into tubules behaved like Akita, while a substrate (type I procollagen) that is degraded by the ER-phagy sheets receptor, FAM134B, did not. Conversely, when ER tubules were augmented in Lunapark knock-out cells by overexpressing reticulons, ER-phagy increased and the number of large Akita puncta was reduced. Our findings imply that segregating cargoes into tubules has two beneficial roles. First, it localizes mutant misfolded proteins, the receptor, and SEC24C to the same ER domain. Second, physically restraining condensates within tubules, before they undergo ER-phagy, prevents them from enlarging and impacting cell health.

High-content imaging-based pooled CRISPR screens in mammalian cells.

CRISPR (clustered regularly interspaced short palindromic repeats)-based gene inactivation provides a powerful means for linking genes to particular cellular phenotypes. CRISPR-based screening typically uses large genomic pools of single guide RNAs (sgRNAs). However, this approach is limited to phenotypes that can be enriched by chemical selection or FACS sorting. Here, we developed a microscopy-based approach, which we name optical enrichment, to select cells displaying a particular CRISPR-induced phenotype by automated imaging-based computation, mark them by photoactivation of an expressed photoactivatable fluorescent protein, and then isolate the fluorescent cells using fluorescence-activated cell sorting (FACS). A plugin was developed for the open source software µManager to automate the phenotypic identification and photoactivation of cells, allowing ∼1.5 million individual cells to be screened in 8 h. We used this approach to screen 6,092 sgRNAs targeting 544 genes for their effects on nuclear size regulation and identified 14 bona fide hits. These results present a scalable approach to facilitate imaging-based pooled CRISPR screens.

Titrating gene expression using libraries of systematically attenuated CRISPR guide RNAs.

A lack of tools to precisely control gene expression has limited our ability to evaluate relationships between expression levels and phenotypes. Here, we describe an approach to titrate expression of human genes using CRISPR interference and series of single-guide RNAs (sgRNAs) with systematically modulated activities. We used large-scale measurements across multiple cell models to characterize activities of sgRNAs containing mismatches to their target sites and derived rules governing mismatched sgRNA activity using deep learning. These rules enabled us to synthesize a compact sgRNA library to titrate expression of ~2,400 genes essential for robust cell growth and to construct an in silico sgRNA library spanning the human genome. Staging cells along a continuum of gene expression levels combined with single-cell RNA-seq readout revealed sharp transitions in cellular behaviors at gene-specific expression thresholds. Our work provides a general tool to control gene expression, with applications ranging from tuning biochemical pathways to identifying suppressors for diseases of dysregulated gene expression.

Ceapins block the unfolded protein response sensor ATF6α by inducing a neomorphic inter-organelle tether.

The unfolded protein response (UPR) detects and restores deficits in the endoplasmic reticulum (ER) protein folding capacity. Ceapins specifically inhibit the UPR sensor ATF6α, an ER-tethered transcription factor, by retaining it at the ER through an unknown mechanism. Our genome-wide CRISPR interference (CRISPRi) screen reveals that Ceapins function is completely dependent on the ABCD3 peroxisomal transporter. Proteomics studies establish that ABCD3 physically associates with ER-resident ATF6α in cells and in vitro in a Ceapin-dependent manner. Ceapins induce the neomorphic association of ER and peroxisomes by directly tethering the cytosolic domain of ATF6α to ABCD3's transmembrane regions without inhibiting or depending on ABCD3 transporter activity. Thus, our studies reveal that Ceapins function by chemical-induced misdirection which explains their remarkable specificity and opens up new mechanistic routes for drug development and synthetic biology.