Genetic Screens In Mammalian Cells Research Articles

Genetic screening for anticancer genes highlights FBLN5 as a synthetic lethal partner of MYC

BackgroundWhen ectopically overexpressed, anticancer genes, such as TRAIL, PAR4 and ORCTL3, specifically destroy tumour cells without harming untransformed cells. Anticancer genes can not only serve as powerful tumour specific therapy tools but studying their mode of action can reveal mechanisms underlying the neoplastic transformation, sustenance and spread.MethodsAnticancer gene discovery is normally accidental. Here we describe a systematic, gain of function, forward genetic screen in mammalian cells to isolate novel anticancer genes of human origin. Continuing with over 30,000 transcripts from our previous study, 377 cell death inducing genes were subjected to screening. FBLN5 was chosen, as a proof of principle, for mechanistic gene expression profiling, comparison pathways analyses and functional studies.ResultsSixteen novel anticancer genes were isolated; these included non-coding RNAs, protein-coding genes and novel transcripts, such as ZNF436-AS1, SMLR1, TMEFF2, LINC01529, HYAL2, NEIL2, FBLN5, YPEL4 and PHKA2-processed transcript. FBLN5 selectively caused inhibition of MYC in COS-7 (transformed) cells but not in CV-1 (normal) cells. MYC was identified as synthetic lethality partner of FBLN5 where MYC transformed CV-1 cells experienced cell death upon FBLN5 transfection, whereas FBLN5 lost cell death induction in MCF-7 cells upon MYC knockdown.ConclusionsSixteen novel anticancer genes are present in human genome including FBLN5. MYC is a synthetic lethality partner of FBLN5.-ju642ik93t1hmw-_xNSRiVideo

New regulators of the tetracycline-inducible gene expression system identified by chemical and genetic screens.

The tetracycline repressor (tetR)‐regulated system is a widely used tool to specifically control gene expression in mammalian cells. Based on this system, we generated a human osteosarcoma cell line, which allows for the inducible expression of an EGFP fusion of the TAR DNA‐binding protein 43 (TDP‐43), which has been linked to neurodegenerative diseases. Consistent with previous findings, TDP‐43 overexpression led to the accumulation of aggregates and limited the viability of U2OS. Using this inducible system, we conducted a chemical screen with a library that included FDA‐approved drugs. While the primary screen identified several compounds that prevented TDP‐43 toxicity, further experiments revealed that these chemicals abrogated the doxycycline‐dependent TDP‐43 expression. This antagonistic effect was observed with both doxycycline and tetracycline, and in several Tet‐On cell lines expressing different genes, confirming the general effect of these compounds as inhibitors of the tetR system. Using the same cell line, a genome‐wide CRISPR/Cas9 screen identified epigenetic regulators such as the G9a methyltransferase and TRIM28 as potential modifiers of TDP‐43 toxicity. Yet again, further experiments revealed that G9a inhibition or TRIM28 loss prevented doxycycline‐dependent expression of TDP‐43. In summary, we have identified new chemical and genetic regulators of the tetR system, thereby raising awareness of the limitations of this approach to conduct chemical or genetic screening in mammalian cells.

CRISPR Interference (CRISPRi) and CRISPR Activation (CRISPRa) to Explore the Oncogenic lncRNA Network.

The human genome contains thousands of long noncoding RNAs (lncRNAs), even outnumbering protein-coding genes. These molecules can play a pivotal role in the development and progression of human disease, including cancer, and are susceptible to therapeutic intervention. Evidence of biologic function, however, is still missing for the vast majority of them. Both loss-of-function (LOF) and gain-of-function (GOF) studies are therefore necessary to advance our understanding of lncRNA networks and programs driving tumorigenesis. Here, we describe a protocol to perform lncRNA's LOF or GOF studies in multiple myeloma (MM) cells, using CRISPR interference (CRISPRi) or CRISPR activation (CRISPRa) technologies, respectively. These approaches have many advantages, including applicability to large-scale genetic screens in mammalian cells and possible reversibility of modulating effects; moreover, CRISPRa offers the unique opportunity to enhance lncRNA expression at the site of transcription, with relevant biologic implications.

Combined lentiviral- and RNA-mediated CRISPR/Cas9 delivery for efficient and traceable gene editing in human hematopoietic stem and progenitor cells

The CRISPR/Cas9 system is a versatile tool for functional genomics and forward genetic screens in mammalian cells. However, it has been challenging to deliver the CRISPR components to sensitive cell types, such as primary human hematopoietic stem and progenitor cells (HSPCs), partly due to lentiviral transduction of Cas9 being extremely inefficient in these cells. Here, to overcome these hurdles, we developed a combinatorial system using stable lentiviral delivery of single guide RNA (sgRNA) followed by transient transfection of Cas9 mRNA by electroporation in human cord blood-derived CD34+ HSPCs. We further applied an optimized sgRNA structure, that significantly improved editing efficiency in this context, and we obtained knockout levels reaching 90% for the cell surface proteins CD45 and CD44 in sgRNA transduced HSPCs. Our combinatorial CRISPR/Cas9 delivery approach had no negative influence on CD34 expression or colony forming capacity in vitro compared to non-treated HSPCs. Furthermore, gene edited HSPCs showed intact in vivo reconstitution capacity following transplantation to immunodeficient mice. Taken together, we developed a paradigm for combinatorial CRISPR/Cas9 delivery that enables efficient and traceable gene editing in primary human HSPCs, and is compatible with high functionality both in vitro and in vivo.

Identification of the HECT E3 ligase UBR5 as a regulator of MYC degradation using a CRISPR/Cas9 screen

MYC oncoprotein is a multifunctional transcription factor that regulates the expression of a large number of genes involved in cellular growth, proliferation and metabolism. Altered MYC protein level lead to cellular transformation and tumorigenesis. MYC is deregulated in > 50% of human cancers, rendering it an attractive drug target. However, direct inhibition of this class of proteins using conventional small molecules is challenging due to their intrinsically disordered state. To discover novel posttranslational regulators of MYC protein stability and turnover, we established a genetic screen in mammalian cells by combining a fluorescent protein-based MYC abundance sensor, CRISPR/Cas9-based gene knockouts and next-generation sequencing. Our screen identifies UBR5, an E3 ligase of the HECT-type family, as a novel regulator of MYC degradation. Even in the presence of the well-described and functional MYC ligase, FBXW7, UBR5 depletion leads to accumulation of MYC in cells. We demonstrate interaction of UBR5 with MYC and reduced K48-linked ubiquitination of MYC upon loss of UBR5 in cells. Interestingly, in cancer cell lines with amplified MYC expression, depletion of UBR5 resulted in reduced cell survival, as a consequence of MYC stabilization. Finally, we show that MYC and UBR5 are co-amplified in more than 40% of cancer cells and that MYC copy number amplification correlates with enhanced transcriptional output of UBR5. This suggests that UBR5 acts as a buffer in MYC amplified settings and protects these cells from apoptosis.

Abstract IA08: Pooled CRISPR screens for novel autophagy-related factors identify TMEM41, an ER-resident protein predicted to be a channel

Abstract The power of forward genetics in yeast is the foundation on which the field of autophagy research firmly stands. Complementary work on autophagy in metazoans has revealed both the deep conservation of this process, as well as novel mechanisms by which autophagy is regulated in the context of development, immunity, and neuronal homeostasis. The recent emergence of new CRISPR/Cas9-based technologies has begun facilitating efforts to define novel autophagy factors and pathways by forward genetic screening in mammalian cells. Here, we set out to develop an expanded and improved toolkit of autophagy reporters for CRISPR/Cas9 screening. Genome-wide screening of our reporters in mammalian cells recovered virtually all known ATG factors as well as previously uncharacterized factors including VPS37A, TMEM251, ALS2, ACP1, and TMEM41B. To validate this dataset, we used quantitative microscopy and biochemical analyses to show that one novel hit, TMEM41B, is required for a late step in phagophore maturation. We found that TMEM41B is an integral ER membrane protein distantly related to the established autophagy factor VMP1, and our data show that these two factors play related, albeit not fully overlapping, roles in autophagosome biogenesis. In sum, our work uncovers new ATG factors, reveals a malleable network of autophagy receptor genetic interactions, and provides a valuable resource (http://crispr.deniclab.com) for further mining of novel autophagy mechanisms. Citation Format: Vladimir Denic. Pooled CRISPR screens for novel autophagy-related factors identify TMEM41, an ER-resident protein predicted to be a channel [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr IA08.

Pooled analysis of radiation hybrids identifies loci for growth and drug action in mammalian cells.

Genetic screens in mammalian cells commonly focus on loss-of-function approaches. To evaluate the phenotypic consequences of extra gene copies, we used bulk segregant analysis (BSA) of radiation hybrid (RH) cells. We constructed six pools of RH cells, each consisting of ∼2500 independent clones, and placed the pools under selection in media with or without paclitaxel. Low pass sequencing identified 859 growth loci, 38 paclitaxel loci, 62 interaction loci, and three loci for mitochondrial abundance at genome-wide significance. Resolution was measured as ∼30 kb, close to single-gene. Divergent properties were displayed by the RH-BSA growth genes compared to those from loss-of-function screens, refuting the balance hypothesis. In addition, enhanced retention of human centromeres in the RH pools suggests a new approach to functional dissection of these chromosomal elements. Pooled analysis of RH cells showed high power and resolution and should be a useful addition to the mammalian genetic toolkit.

Optical Pooled Screens in Human Cells

Genetic screens are critical for the systematic identification of genes underlying cellular phenotypes. Pooling gene perturbations greatly improves scalability but is not compatible with imaging of complex and dynamic cellular phenotypes. Here, we introduce a pooled approach for optical genetic screens in mammalian cells. We use targeted in situ sequencing to demultiplex a library of genetic perturbations following image-based phenotyping. We screened a set of 952 genes across millions of cells for involvement in nuclear factor κB (NF-κB) signaling by imaging the translocation of RelA (p65) to the nucleus. Screening at a single time point across 3 cell lines recovered 15 known pathway components, while repeating the screen with live-cell imaging revealed a role for Mediator complex subunits in regulating the duration of p65 nuclear retention. These results establish a highly multiplexed approach to image-based screens of spatially and temporally defined phenotypes with pooled libraries.

Pooled CRISPR-Based Genetic Screens in Mammalian Cells.

Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian cells, this technology represents a novel means to perform genome-wide genetic screens for functional genomics studies. Libraries of guide RNAs (sgRNA) targeting all open reading frames permit the facile generation of thousands of genetic perturbations in a single pool of cells that can be screened for specific phenotypes to implicate gene function and cellular processes in an unbiased and systematic way. CRISPR-Cas screens provide researchers with a simple, efficient, and inexpensive method to uncover the genetic blueprints for cellular phenotypes. Furthermore, differential analysis of screens performed in various cell lines and from different cancer types can identify genes that are contextually essential in tumor cells, revealing potential targets for specific anticancer therapies. Performing genome-wide screens in human cells can be daunting, as this involves the handling of tens of millions of cells and requires analysis of large sets of data. The details of these screens, such as cell line characterization, CRISPR library considerations, and understanding the limitations and capabilities of CRISPR technology during analysis, are often overlooked. Provided here is a detailed protocol for the successful performance of pooled genome-wide CRISPR-Cas9 based screens.

Pooled CRISPR-Based Genetic Screens in Mammalian Cells

Pooled CRISPR-Based Genetic Screens in Mammalian Cells

COMBINED LENTIVIRAL- AND RNA-MEDIATED CRISPR/CAS9 DELIVERY FOR EFFICIENT AND TRACEABLE GENE EDITING IN HUMAN HEMATOPOIETIC STEM AND PROGENITOR CELLS

The CRISPR/Cas9 system has been highlighted as an ideal tool for large-scale forward genetic screens in mammalian cells. To deliver CRISPR libraries by lentiviral transduction is advantageous in a pooled screen context where an integrated and traceable component is required. However, lentiviral delivery of the CRISPR components to primary human hematopoietic stem and progenitor cells (HSPCs) has proven challenging and limited the screening opportunities in these cells. Here, we addressed these challenges by developing a hybrid system combining stable delivery of single guide RNA (sgRNA) by lentiviral transduction, with transient delivery of Cas9 by electroporation. We optimized and compared delivery modes of Cas9 in cord blood-derived CD34+ HSPCs transduced with lentiviral sgRNAs targeting the gene PTPRC (CD45), and delivered Cas9 either as mRNA or protein by electroporation to assess gene knockout in terms of loss of CD45 expression evaluated by flow cytometry. We found that Cas9 mRNA is preferable to Cas9 protein in terms of editing efficiency and acceptable cytotoxicity. We further implemented an optimized sgRNA structure, which significantly improved the editing efficiency, reaching 90% knockout of CD45 in GFP+ sgRNA transduced cells. Importantly, this was also observed for several sgRNAs targeting CD29 and CD44, reaching editing efficiencies such as 62% and 88% compared to 30% and 76%, respectively. This gene editing did not affect CD34 expression or in vitro colony forming capacity compared to untreated or mock treated cells. Furthermore, transplantation experiments in immunodeficient mice showed that the gene edited HSPCs retained efficient reconstitution capacity in vivo. Taken together, this hybrid system not only balances efficient editing with low cytotoxicity, but also enables cost-effective, traceable gene editing for screening purposes in human HSPCs.

Engineering Forward Genetics into Cultured Cancer Cells for Chemical Target Identification

Target identification for biologically active small molecules remains a major barrier for drug discovery. Cancer cells exhibiting defective DNA mismatch repair (dMMR) have been used as a forward genetics system to uncover compound targets. However, thisapproach has been limited by the dearth of cancer cell lines that harbor naturally arising dMMR. Here,we establish a platform for forward genetic screening using CRISPR/Cas9 to engineer dMMR into mammalian cells. We demonstrate the utility of this approach to identify mechanisms of drug action in mouse and human cancer cell lines using invitro selections against three cellular toxins. In each screen, compound-resistant alleles emerged in drug-resistant clones, supporting the notion that engineered dMMR enables forward genetic screening in mammalian cells.

CRISPR screening using an expanded toolkit of autophagy reporters identifies TMEM41B as a novel autophagy factor.

The power of forward genetics in yeast is the foundation on which the field of autophagy research firmly stands. Complementary work on autophagy in higher eukaryotes has revealed both the deep conservation of this process, as well as novel mechanisms by which autophagy is regulated in the context of development, immunity, and neuronal homeostasis. The recent emergence of new clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-based technologies has begun facilitating efforts to define novel autophagy factors and pathways by forward genetic screening in mammalian cells. Here, we set out to develop an expanded toolkit of autophagy reporters amenable to CRISPR/Cas9 screening. Genome-wide screening of our reporters in mammalian cells recovered virtually all known autophagy-related (ATG) factors as well as previously uncharacterized factors, including vacuolar protein sorting 37 homolog A (VPS37A), transmembrane protein 251 (TMEM251), amyotrophic lateral sclerosis 2 (ALS2), and TMEM41B. To validate this data set, we used quantitative microscopy and biochemical analyses to show that 1 novel hit, TMEM41B, is required for phagophore maturation. TMEM41B is an integral endoplasmic reticulum (ER) membrane protein distantly related to the established autophagy factor vacuole membrane protein 1 (VMP1), and our data show that these two factors play related, albeit not fully overlapping, roles in autophagosome biogenesis. In sum, our work uncovers new ATG factors, reveals a malleable network of autophagy receptor genetic interactions, and provides a valuable resource (http://crispr.deniclab.com) for further mining of novel autophagy mechanisms.

CRISPRi and CRISPRa Screens in Mammalian Cells for Precision Biology and Medicine.

Next-generation DNA sequencing technologies have led to a massive accumulation of genomic and transcriptomic data from patients and healthy individuals. The major challenge ahead is to understand the functional significance of the elements of the human genome and transcriptome, and implications for diagnosis and treatment. Genetic screens in mammalian cells are a powerful approach to systematically elucidating gene function in health and disease states. In particular, recently developed CRISPR/Cas9-based screening approaches have enormous potential to uncover mechanisms and therapeutic strategies for human diseases. The focus of this review is the use of CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) for genetic screens in mammalian cells. We introduce the underlying technology and present different types of CRISPRi/a screens, including those based on cell survival/proliferation, sensitivity to drugs or toxins, fluorescent reporters, and single-cell transcriptomes. Combinatorial screens, in which large numbers of gene pairs are targeted to construct genetic interaction maps, reveal pathway relationships and protein complexes. We compare and contrast CRISPRi and CRISPRa with alternative technologies, including RNA interference (RNAi) and CRISPR nuclease-based screens. Finally, we highlight challenges and opportunities ahead.

CRISPR/Cas9 Genome-Editing System in Human Stem Cells: Current Status and Future Prospects.

Genome-editing involves the insertion, deletion, or replacement of DNA in the genome of a living organism using “molecular scissors.” Traditional genome editing with engineered nucleases for human stem cells is limited by its low efficiency, high cost, and poor specificity. The CRISPR system has recently emerged as a powerful gene manipulation technique with advantages of high editing efficiency and low cost. Although this technique offers huge potential for gene manipulation in various organisms ranging from prokaryotes to higher mammals, there remain many challenges in human stem cell research. In this review, we highlight the basic biology and application of the CRISPR/Cas9 system in current human stem cell research, discuss its advantages and challenges, and debate the future prospects for human stem cells in regenerative medicine.

Elucidating drug targets and mechanisms of action by genetic screens in mammalian cells.

Phenotypic screening is a powerful approach to discover small molecules with desired effects on biological systems, which can then be developed into therapeutic drugs. The identification of the target and mechanism of action of compounds discovered in phenotypic screens remains a major challenge. This feature article describes the use of genetic tools to reveal drug targets and mechanisms in mammalian cells. Until recently, RNA interference was the method of choice for such studies. Here, we highlight very recent additions to the genetic toolkit in mammalian cells, including CRISPR, CRISPR interference, and CRISPR activation, and illustrate their usefulness for drug target identification.

Genomic Copy Number Dictates a Gene-Independent Cell Response to CRISPR/Cas9 Targeting.

The CRISPR/Cas9 system enables genome editing and somatic cell genetic screens in mammalian cells. We performed genome-scale loss-of-function screens in 33 cancer cell lines to identify genes essential for proliferation/survival and found a strong correlation between increased gene copy number and decreased cell viability after genome editing. Within regions of copy-number gain, CRISPR/Cas9 targeting of both expressed and unexpressed genes, as well as intergenic loci, led to significantly decreased cell proliferation through induction of a G2 cell-cycle arrest. By examining single-guide RNAs that map to multiple genomic sites, we found that this cell response to CRISPR/Cas9 editing correlated strongly with the number of target loci. These observations indicate that genome targeting by CRISPR/Cas9 elicits a gene-independent antiproliferative cell response. This effect has important practical implications for the interpretation of CRISPR/Cas9 screening data and confounds the use of this technology for the identification of essential genes in amplified regions. We found that the number of CRISPR/Cas9-induced DNA breaks dictates a gene-independent antiproliferative response in cells. These observations have practical implications for using CRISPR/Cas9 to interrogate cancer gene function and illustrate that cancer cells are highly sensitive to site-specific DNA damage, which may provide a path to novel therapeutic strategies. Cancer Discov; 6(8); 914-29. ©2016 AACR.See related commentary by Sheel and Xue, p. 824See related article by Munoz et al., p. 900This article is highlighted in the In This Issue feature, p. 803.

Large-Scale Single Guide RNA Library Construction and Use for CRISPR-Cas9-Based Genetic Screens.

The ability to systematically disrupt genes serves as a powerful tool for understanding their function. The programmable CRISPR-Cas9 system enables efficient targeting of large numbers of genes through the use of single guide RNA (sgRNA) libraries. In cultured mammalian cells, collections of knockout mutants can be readily generated by means of transduction of Cas9-sgRNA lentiviral pools, screened for a phenotype of interest, and counted using high-throughput DNA sequencing. This technique represents the first general method for undertaking systematic loss-of-function genetic screens in mammalian cells. Here, we introduce the methodology and rationale for conducting CRISPR-based screens, focusing on distinguishing positive and negative selection strategies.

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice — A High-throughput Screen in ES Cells

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely limits its potential. We describe here the use of mouse embryonic stem (ES) cells as surrogate cells to screen for a phenotype of interest and subsequently introduce these cells into a host embryo to develop into a living mouse carrying the phenotype. This method provides (1) a cost effective, high-throughput platform for genetic screen in mammalian cells; (2) a rapid way to identify the mutated genes and verify causality; and (3) a short-cut to develop mouse mutants directly from these selected ES cells for whole animal studies. We demonstrated the use of paraquat (PQ) to select resistant mutants and identify mutations that confer oxidative stress resistance. Other stressors or cytotoxic compounds may also be used to screen for resistant mutants to uncover novel genetic determinants of a variety of cellular stress resistance.

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice - A High-throughput Screen in ES Cells.

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely limits its potential. We describe here the use of mouse embryonic stem (ES) cells as surrogate cells to screen for a phenotype of interest and subsequently introduce these cells into a host embryo to develop into a living mouse carrying the phenotype. This method provides (1) a cost effective, high-throughput platform for genetic screen in mammalian cells; (2) a rapid way to identify the mutated genes and verify causality; and (3) a short-cut to develop mouse mutants directly from these selected ES cells for whole animal studies. We demonstrated the use of paraquat (PQ) to select resistant mutants and identify mutations that confer oxidative stress resistance. Other stressors or cytotoxic compounds may also be used to screen for resistant mutants to uncover novel genetic determinants of a variety of cellular stress resistance.