Dual gene activation and knockout screen reveals directional dependencies in genetic networks

Boettcher, Michael; Tian, Ruilin; Blau, James; Markegard, Evan; Wagner, Ryan T.; Wu, David; Mo, Xiulei; Biton, Anne; Zaitlen, Noah; Fu, Haian; McCormick, Frank; Kampmann, Martin; McManus, Michael T.

doi:10.1038/nbt.4062

Cited by 125 publications

(103 citation statements)

References 52 publications

(58 reference statements)

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“…PVT1 was previously shown to be a transacting lncRNA that stabilizes the MYC protein, promoting oncogenesis [110]. Consistent with its known tumor-promoting properties, CRISPRa-mediated activation of PVT1 conferred drug resistance to leukemia cells, as shown through pooled genetic screens [107,108]. Unexpectedly, CRISPRi-mediated repression of PVT1 also enhanced the proliferation of leukemia and breast cancer cells [18].…”

Section: Reconciling Lessons Learned From Perturbation Of Lncrna Exprmentioning

confidence: 79%

Modulating the expression of long non‐coding RNA s for functional studies

Liu

Lim

2018

EMBO Reports

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Long non‐coding RNAs (lncRNAs) have emerged as important regulators of cell biology. The mechanisms by which lncRNAs function are likely numerous, and most are poorly understood. Currently, the mechanisms of functional lncRNAs include those that directly involve the lncRNA transcript, the process of their own transcription and splicing, and even underlying transcriptional regulatory elements within the genomic DNA that encodes the lncRNA. As our understanding of lncRNA biology evolves, so have the methods that are utilized to elucidate their functions. In this review, we survey a collection of different methods used to modulate lncRNA expression levels for the assessment of biological function. From RNA‐targeted strategies, genetic deletions, to engineered gene regulatory systems, the advantages and caveats of each method will be discussed. Ultimately, the selection of tools will be guided by which potential lncRNA mechanisms are being investigated, and no single method alone will likely be sufficient to reveal the function of any particular lncRNA.

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Section: Reconciling Lessons Learned From Perturbation Of Lncrna Exprmentioning

confidence: 79%

Modulating the expression of long non‐coding RNA s for functional studies

Liu

Lim

2018

EMBO Reports

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“…More recently, hallmark papers in S. cerevisiae revealed the first and only comprehensive functional genetic landscape of a cell (Costanzo et al, 2010, 2016). Additionally, GI mapping efforts in prokaryotes, as well recent work in S. pombe, fruit fly and human cells, demonstrate the general utility and enormous promise of GI maps across diverse organisms (Babu et al, 2014; Bassik et al, 2013; Boettcher et al, 2018; Du et al, 2017; Fischer et al, 2015; Frost et al, 2012; Han et al, 2017; Roguev et al, 2007, 2013; Rosenbluh et al, 2016; Shen et al, 2017; Wong et al, 2016). …”

Section: Introductionmentioning

confidence: 98%

Mapping the Genetic Landscape of Human Cells

Horlbeck

Wang

et al. 2018

Cell

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251

241

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Seminal yeast studies have established the value of comprehensively mapping genetic interactions (GIs) for inferring gene function. Efforts in human cells using focused gene sets underscore the utility of this approach, but the feasibility of generating large-scale, diverse human GI maps remains unresolved. We developed a CRISPR interference platform for large-scale quantitative mapping of human GIs. We systematically perturbed 222,784 gene pairs in two cancer cell lines. The resultant maps cluster functionally related genes, assigning function to poorly characterized genes, including TMEM261, a new electron transport chain component. Individual GIs pinpoint unexpected relationships between pathways, exemplified by a specific cholesterol biosynthesis intermediate whose accumulation induces deoxynucleotide depletion, causing replicative DNA damage and a synthetic-lethal interaction with the ATR/9-1-1 DNA repair pathway. Our map provides a broad resource, establishes GI maps as a high-resolution tool for dissecting gene function, and serves as a blueprint for mapping the genetic landscape of human cells.

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“…However, the generation of such datasets faces major limitations. In addition to the roughly 16-fold increase in combinatorial space of all possible double knockouts in human cells compared to yeast (due to the four-fold increase in genes), human screening libraries for simultaneously knocking out multiple genes of interest are relatively new and still face unsolved technical challenges (Boettcher et al, 2018; Du et al, 2017; Han et al, 2017; Najm et al, 2018; Shen et al, 2017). In contrast, genome-scale single-gene knockout libraries are technically more advanced and have been used extensively in pooled fitness screens (Doench, 2018).…”

Section: Introductionmentioning

confidence: 99%

Interrogation of Mammalian Protein Complex Structure, Function, and Membership Using Genome-Scale Fitness Screens

et al. 2018

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Summary Protein complexes are assemblies of subunits that have co-evolved to execute one or many coordinated functions in the cellular environment. Functional annotation of mammalian protein complexes is critical to understanding biological processes as well as disease mechanisms. Here, we used genetic co-essentiality derived from genome-scale RNAi- and CRISPR-Cas9- based fitness screens performed across hundreds of human cancer cell lines to assign measures of functional similarity. From these measures, we systematically built and characterized functional similarity networks which recapitulate known structural and functional features of well-studied protein complexes and resolve novel functional modules within complexes lacking structural resolution, such as the mammalian SWI/SNF complex. Finally, by integrating functional networks with large protein-protein interaction networks, we discovered novel protein complexes involving recently-evolved genes of unknown function. Taken together, these findings demonstrate the utility of genetic perturbation screens alone and in combination with large-scale biophysical data to enhance our understanding of mammalian protein complexes in normal and disease states.

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Dual gene activation and knockout screen reveals directional dependencies in genetic networks

Cited by 125 publications

References 52 publications

Modulating the expression of long non‐coding RNA s for functional studies

Modulating the expression of long non‐coding RNA s for functional studies

Mapping the Genetic Landscape of Human Cells

Interrogation of Mammalian Protein Complex Structure, Function, and Membership Using Genome-Scale Fitness Screens

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