CRISPR Interference Screens Research Articles

Inhibition by misdirection

Cell Biology The endoplasmic reticulum (ER) is the main entry point to the cellular secretory pathway. It is a major site of folding and quality control of newly synthesized proteins. When protein folding goes awry, the unfolded protein response (UPR) is activated to protect the cell from harmful effects of aberrant proteins. The UPR is triggered by an ER-tethered transcription factor, ATF6α. Small-molecule inhibitors known as Ceapins specifically inhibit ATF6α by retaining it at the ER. Torres et al. performed a genome-wide CRISPR interference screen to elucidate how Ceapins work. They found that Ceapins act via the ABCD3 peroxisomal transporter. In the presence of Ceapins, ABCD3 binds to ATF6α, which means the ER becomes tethered to peroxisomes and prevents ATF6α from leaving the ER and carrying out its function. eLife 8 , e46595 (2019).

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.

332 A noncoding RNA CRISPR-interference screen identifies novel regulators and pathways of human epidermis formation

332 A noncoding RNA CRISPR-interference screen identifies novel regulators and pathways of human epidermis formation

Pooled CRISPR interference screening enables genome-scale functional genomics study in bacteria with superior performance

To fully exploit the microbial genome resources, a high-throughput experimental platform is needed to associate genes with phenotypes at the genome level. We present here a novel method that enables investigation of the cellular consequences of repressing individual transcripts based on the CRISPR interference (CRISPRi) pooled screening in bacteria. We identify rules for guide RNA library design to handle the unique structure of prokaryotic genomes by tiling screening and construct an E. coli genome-scale guide RNA library (~60,000 members) accordingly. We show that CRISPRi outperforms transposon sequencing, the benchmark method in the microbial functional genomics field, when similar library sizes are used or gene length is short. This tool is also effective for mapping phenotypes to non-coding RNAs (ncRNAs), as elucidated by a comprehensive tRNA-fitness map constructed here. Our results establish CRISPRi pooled screening as a powerful tool for mapping complex prokaryotic genetic networks in a precise and high-throughput manner.

Abstract PR13: CRISPRi screening with targeted therapeutics classifies functional long non-coding RNAs in DLBCL

Abstract While tens of thousands of lncRNAs have been detected in the human genome, the functional significance of the majority of these genes remain untested. Here, we use CRISPR interference (CRISPRi) loss-of-function screening combined with targeted drug treatment to identify oncogenic and tumor suppressive lncRNAs. Integrative analysis of 417 primary human tumor and cell line RNA-seq datasets from Diffuse Large B Cell Lymphoma (DLBCL) identified 1,276 intergenic lncRNA candidates, including 59 de novo assembled transcripts. CRISPRi screening in a genetically diverse panel of 4 DLBCL cell lines identified known ABC DLBCL protein-coding oncogenes (BTK, CD79A) and demonstrated oncogenic function of 247 lncRNAs including MIR155HG and MALAT1. To assign functionally-related pathways to unknown lncRNAs, we performed this CRISPRi screen in the context of drugs with known targets: Ibrutinib (BTK), JQ1 (BRD4), and Etoposide (TOP2A). Significant synergy or resistance to drug treatment was observed with 54 lncRNAs. This ongoing work demonstrates a high-throughput pipeline for functional classification of lncRNAs using synthetic lethality screening in cancer. Citation Format: Dan E. Webster, James D. Phelan, Monica Kasbekar, Arthur L. Shaffer, III, Louis M. Staudt. CRISPRi screening with targeted therapeutics classifies functional long non-coding RNAs in DLBCL [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr PR13.

Genetic interaction mapping in mammalian cells using CRISPR interference.

We describe a combinatorial CRISPR interference (CRISPRi) screening platform for mapping genetic interactions in mammalian cells. We targeted 107 chromatin-regulation factors in human cells with pools of either single or double single guide RNAs (sgRNAs) to downregulate individual genes or gene pairs, respectively. Relative enrichment analysis of individual sgRNAs or sgRNA pairs allowed for quantitative characterization of genetic interactions, and comparison with protein-protein-interaction data revealed a functional map of chromatin regulation.

A Multiplexed Single-Cell CRISPR Screening Platform Enables Systematic Dissection of the Unfolded Protein Response

Functional genomics efforts face tradeoffs between number of perturbations examined and complexity of phenotypes measured. We bridge this gap with Perturb-seq, which combines droplet-based single-cell RNA-seq with a strategy for barcoding CRISPR-mediated perturbations, allowing many perturbations to be profiled in pooled format. We applied Perturb-seq to dissect the mammalian unfolded protein response (UPR) using single and combinatorial CRISPR perturbations. Two genome-scale CRISPR interference (CRISPRi) screens identified genes whose repression perturbs ER homeostasis. Subjecting ∼100 hits to Perturb-seq enabled high-precision functional clustering of genes. Single-cell analyses decoupled the three UPR branches, revealed bifurcated UPR branch activation among cellssubject to the same perturbation, and uncovered differential activation of the branches across hits, including an isolated feedback loop between the translocon and IRE1α. These studies provide insight into how the three sensors of ER homeostasis monitor distinct types of stress and highlight the ability of Perturb-seq to dissect complex cellular responses.

Quantitative CRISPR interference screens in yeast identify chemical-genetic interactions and new rules for guide RNA design.

BackgroundGenome-scale CRISPR interference (CRISPRi) has been used in human cell lines; however, the features of effective guide RNAs (gRNAs) in different organisms have not been well characterized. Here, we define rules that determine gRNA effectiveness for transcriptional repression in Saccharomyces cerevisiae.ResultsWe create an inducible single plasmid CRISPRi system for gene repression in yeast, and use it to analyze fitness effects of gRNAs under 18 small molecule treatments. Our approach correctly identifies previously described chemical-genetic interactions, as well as a new mechanism of suppressing fluconazole toxicity by repression of the ERG25 gene. Assessment of multiple target loci across treatments using gRNA libraries allows us to determine generalizable features associated with gRNA efficacy. Guides that target regions with low nucleosome occupancy and high chromatin accessibility are clearly more effective. We also find that the best region to target gRNAs is between the transcription start site (TSS) and 200 bp upstream of the TSS. Finally, unlike nuclease-proficient Cas9 in human cells, the specificity of truncated gRNAs (18 nt of complementarity to the target) is not clearly superior to full-length gRNAs (20 nt of complementarity), as truncated gRNAs are generally less potent against both mismatched and perfectly matched targets.ConclusionsOur results establish a powerful functional and chemical genomics screening method and provide guidelines for designing effective gRNAs, which consider chromatin state and position relative to the target gene TSS. These findings will enable effective library design and genome-wide programmable gene repression in many genetic backgrounds.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-0900-9) contains supplementary material, which is available to authorized users.