Genome-wide CRISPR screens using isogenic cells reveal vulnerabilities conferred by loss of tumor suppressors

Feng, Xu; Tang, Mengfan; Dede, Merve; Su, Dan; Pei, Guangsheng; Jiang, Dadi; Wang, Chao; Chen, Zhen; Li, Mi; Nie, Litong; Xiong, Yun; Li, Siting; Park, Jeong-Min; Zhang, Huimin; Huang, Min; Szymonowicz, Klaudia; Zhao, Zhongming; Hart, Traver; Chen, Junjie

doi:10.1126/sciadv.abm6638

Cited by 24 publications

(21 citation statements)

References 71 publications

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“…However, the dual-guide strategy offers several practical advantages over existing genetic modifier screening strategies. Our approach eliminates the need to create and characterize a knock-out line for a particular gene of interest (Hickey et al, 2020; Feng et al, 2022; Westermann et al, 2022; Fu et al, 2013; Rossi et al, 2015). It also allows for the simultaneous delivery and selection of both targeted and genome-wide elements, resulting in less cell line construction and manipulation.…”

Section: Discussionmentioning

confidence: 99%

A dual sgRNA library design to probe genetic modifiers using genome-wide CRISPRi screens

Guna

Page

Replogle

et al. 2023

Preprint

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The ability to map genetic interactions has been essential for determining gene function and defining biological pathways. Therefore, a system to readily perform genome-wide genetic modifier screens in human cells is a powerful platform for dissecting complex processes in mammalian cells, where redundancy and adaptation commonly mask the phenotype of a single genetic perturbation. Here, we report a CRISPR interference (CRISPRi) based platform, compatible with Fluorescence Activated Cell Sorting (FACS)-based reporter screens, that can be used to query epistatic relationships at scale. This is enabled by a flexible dual-sgRNA library design that allows for the simultaneous delivery and selection of a fixed sgRNA and a second randomized guide, comprised of a genome-wide library, with a single transduction. As a proof of principle, we apply our approach to study the pathways that mediate tail-anchored (TA) protein insertion at the endoplasmic reticulum (ER). We show that this dual-guide library approach can be successfully coupled with FACS-based reporter screening, to identify genetic epistasis and thereby place TA biogenesis factors in their respective parallel pathways. We demonstrate that this dual-guide approach is both more sensitive and specific than traditional growth screening approaches, and is ideally suited for dissecting the complex interplay between factors in human cells.

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Section: Discussionmentioning

confidence: 99%

A dual sgRNA library design to probe genetic modifiers using genome-wide CRISPRi screens

Guna

Page

Replogle

et al. 2023

Preprint

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“…The CRISPR screens, using the Toronto Knock Out Library v3 (TKOv3) gRNA library, were conducted as described previously [ 31 , 32 ]. Briefly, 120 million HEK293A WT cells and 200 million ZATT KO cells were infected with the TKOv3 library lentiviruses at a low MOI (<0.3).…”

Section: Methodsmentioning

confidence: 99%

Genome-Wide CRISPR Screens Reveal ZATT as a Synthetic Lethal Target of TOP2-Poison Etoposide That Can Act in a TDP2-Independent Pathway

Park

Zhang

Nie

et al. 2023

IJMS

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Etoposide (ETO) is an anticancer drug that targets topoisomerase II (TOP2). It stabilizes a normally transient TOP2–DNA covalent complex (TOP2cc), thus leading to DNA double-strand breaks (DSBs). Tyrosyl-DNA phosphodiesterases two (TDP2) is directly involved in the repair of TOP2cc by removing phosphotyrosyl peptides from 5′-termini of DSBs. Recent studies suggest that additional factors are required for TOP2cc repair, which include the proteasome and the zinc finger protein associated with TDP2 and TOP2, named ZATT. ZATT may alter the conformation of TOP2cc in a way that renders the accessibility of TDP2 for TOP2cc removal. In this study, our genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screens revealed that ZATT also has a TDP2-independent role in promoting cell survival following ETO treatment. ZATT KO cells showed relatively higher ETO sensitivity than TDP2-KO cells, and ZATT/TDP2 DKO cells displayed additive hypersensitivity to ETO treatment. The study using a series of deletion mutants of ZATT determined that the N-terminal 1–168 residues of ZATT are required for interaction with TOP2 and this interaction is critical to ETO sensitivity. Moreover, depletion of ZATT resulted in accelerated TOP2 degradation after ETO or cycloheximide (CHX) treatment, suggesting that ZATT may increase TOP2 stability and likely participate in TOP2 turnover. Taken together, this study suggests that ZATT is a critical determinant that dictates responses to ETO treatment and targeting. ZATT is a promising strategy to increase ETO efficacy for cancer therapy.

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“…One further interesting link between FBXL4 and VHL has been made in a whole genome CRISPR screen for synthetic lethality with common tumour suppressors. Loss of FBXL4 is synthetically lethal with loss of VHL, which based on our findings, we speculate is due to super-elevated levels of BNIP3 and/or NIX (Feng et al , 2022).…”

Section: Discussionmentioning

confidence: 55%

FBXL4 deficiency promotes mitophagy by elevating NIX

Elcocks

Brazel

McCarron

et al. 2022

Preprint

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The selective autophagy of mitochondria is linked to mitochondrial quality control and is critical to a healthy organism. Ubiquitylation is sometimes needed for marking damaged mitochondria for disposal but also for governing the expression and turnover of critical regulatory proteins. We have conducted a CRISPR/Cas9 screen of human E3 ubiquitin ligases for influence on mitophagy under both basal cell culture conditions and following acute mitochondrial depolarisation. We identify two Cullin RING ligases, VHL and FBXL4 as the most profound negative regulators of basal mitophagy. Here we show that these converge through control of the mitophagy adaptors BNIP3 and BNIP3L/NIX, but that this is achieved through different mechanisms. FBXL4 suppression of BNIP3 and NIX levels is mediated via direct interaction and protein destabilisation rather than suppression of HIF1α-mediated transcription. Depletion of NIX but not BNIP3 is sufficient to restore mitophagy levels. Our study enables a full understanding of the aetiology of early onset mitochondrial encephalomyopathy that is supported by analysis of a disease associated mutation. We further show that the compound MLN4924, which globally interferes with Cullin RING ligase activity, is a strong inducer of mitophagy which can provide a research tool in this context as well as a candidate therapeutic agent for conditions linked to mitochondrial quality control.

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Genome-wide CRISPR screens using isogenic cells reveal vulnerabilities conferred by loss of tumor suppressors

Cited by 24 publications

References 71 publications

A dual sgRNA library design to probe genetic modifiers using genome-wide CRISPRi screens

A dual sgRNA library design to probe genetic modifiers using genome-wide CRISPRi screens

Genome-Wide CRISPR Screens Reveal ZATT as a Synthetic Lethal Target of TOP2-Poison Etoposide That Can Act in a TDP2-Independent Pathway

FBXL4 deficiency promotes mitophagy by elevating NIX

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