High Throughput Discovery

Genome-wide CRISPR Screen in a Mouse Model of Tumor Growth and Metastasis

Thu, 03/12/2015 - 00:00
Sidi Chen, Neville E. Sanjana, Kaijie Zheng, Ophir Shalem, Kyungheon Lee, Xi Shi, David A.Scott, Jun Song, Jen Q. Pan, Ralph Weissleder, Hakho Lee, Feng Zhang, and Phillip A. Sharp
Cell
Gabriel Eades
Time
12:00pm
Genetic screens are powerful tools for identifying genes responsible for diverse phenotypes. Here we describe a genome-wide CRISPR/Cas9-mediated loss-of-function screen in tumor growth and metastasis. We mutagenized a non-metastatic mouse cancer cell line using a genome-scale library with 67,405 single-guide RNAs (sgRNAs). The mutant cell pool rapidly generates metastases when transplanted into immunocompromised mice. Enriched sgRNAs in lung metastases and late-stage primary tumors were found to target a small set of genes, suggesting that specific loss-of-function mutations drive tumor growth and metastasis. Individual sgRNAs and a small pool of 624 sgRNAs targeting the top-scoring genes from the primary screen dramatically accelerate metastasis. In all of these experiments, the effect of mutations on primary tumor growth positively correlates with the development of metastases. Our study demonstrates Cas9-based screening as a robust method to systematically assay gene phenotypes in cancer evolution in vivo.
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geades
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Transcriptome in vivo analysis (TIVA) of spatially defined single cells in live tissue

Sat, 02/01/2014 - 00:00
Lovatt D, Ruble BK, Lee J, Dueck H, Kim TK, Fisher S, Francis C, Spaethling JM, Wolf JA, Grady MS, Ulyanova AV, Yeldell SB, Griepenburg JC, Buckley PT, Kim J, Sul JY, Dmochowski IJ, Eberwine J.
Nature Methods
Michael Boettcher
Time
12:00pm
Transcriptome profiling of single cells resident in their natural microenvironment depends upon RNA capture methods that are both noninvasive and spatially precise. We engineered a transcriptome in vivo analysis (TIVA) tag, which upon photoactivation enables mRNA capture from single cells in live tissue. Using the TIVA tag in combination with RNA sequencing (RNA-seq), we analyzed transcriptome variance among single neurons in culture and in mouse and human tissue in vivo. Our data showed that the tissue microenvironment shapes the transcriptomic landscape of individual cells. The TIVA methodology is, to our knowledge, the first noninvasive approach for capturing mRNA from live single cells in their natural microenvironment.
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micboe00
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RNAi screens in mice identify physiological regulators of oncogenic growth.

Wed, 08/14/2013 - 00:00
Beronja S, Janki P, Heller E, Lien WH, Keyes BE, Oshimori N, Fuchs E.
Nature
Junyan Tao
Time
12:00pm
Tissue growth is the multifaceted outcome of a cell’s intrinsic capabilities and its interactions with the surrounding environment. Decoding these complexities is essential for understanding human development and tumorigenesis. Here we tackle this problem by carrying out the first genome-wide RNA-interference-mediated screens in mice. Focusing on skin development and oncogenic (HrasG12V-induced) hyperplasia, our screens uncover previously unknown as well as anticipated regulators of embryonic epidermal growth. Among the top oncogenic screen hits are Mllt6 and the Wnt effector b-catenin, which maintain HrasG12V-dependent hyperproliferation. We also expose b-catenin as an unanticipated antagonist of normal epidermal growth, functioning through Wnt-independent intercellular adhesion. Finally, we validate functional significanceinmouse and human cancers, thereby establishing the feasibility ofin vivomammalian genome-wide investigations to dissect tissue development and tumorigenesis. By documenting some oncogenic growth regulators, we pave the way for future investigations of other hits and raise promise for unearthing new targets for cancer therapies.
 
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chinabamboo
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A nuclear Argonaute promotes multigenerational epigenetic inheritance and germline immortality

Thu, 09/20/2012 - 00:00
Buckley BA, Burkhart KB, Gu SG, Spracklin G, Kershner A, Fritz H, Kimble J, Fire A, Kennedy S.
Nature
Robert Judson
Time
12:00pm
Epigenetic information is frequently erased near the start of each new generation1 . In some cases, however, epigenetic information can be transmitted from parent to progeny (multigenerational epigenetic inheritance)2 . A particularly notable example of this type of epigenetic inheritance is double-stranded RNA-mediated gene silencing in Caenorhabditis elegans. This RNA-mediated interference (RNAi) can be inherited for more than five generations3–8. To understand this process, here we conduct a genetic screen for nematodes defective in transmitting RNAi silencing signals to future generations. This screen identified the heritable RNAi defective 1 (hrde-1) gene. hrde-1 encodes an Argonaute protein that associates with small interfering RNAs in the germ cells of progeny of animals exposed to double-stranded RNA. In the nuclei of these germ cells, HRDE-1 engages the nuclear RNAi defective pathway to direct the trimethylation of histone H3 at Lys 9 (H3K9me3) at RNAi-targeted genomic loci and promote RNAi inheritance. Under normal growth conditions, HRDE-1 associates with endogenously expressed short interfering RNAs, which direct nuclear gene silencing in germ cells. In hrde-1- or nuclear RNAi-deficient animals, germline silencing is lost over generational time. Concurrently, these animals exhibit steadily worsening defects in gamete formation and function that ultimately lead to sterility. These results establish that the Argonaute protein HRDE-1 directs gene-silencing events in germ-cell nuclei that drive multigenerational RNAi inheritance and promote immortality of the germ-cell lineage. We propose that C. elegans use the RNAi inheritance machinery to transmit epigenetic information, accrued by past generations, into future generations to regulate important biological processes.
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Transcriptome-wide Analysis of Regulatory Interactions of the RNA-Binding Protein HuR.

Wed, 06/29/2011 - 00:00
Lebedeva S, Jens M, Theil K, Schwanhäusser B, Selbach M, Landthaler M, Rajewsky N
Mol Cell
https://pubmed.ncbi.nlm.nih.gov/21723171/
Lukas Jeker
Time
12:00pm

Posttranscriptional gene regulation relies on hundreds of RNA binding proteins (RBPs) but the function of most RBPs is unknown. The human RBP HuR/ELAVL1 is a conserved mRNA stability regulator. We used PAR-CLIP, a recently developed method based on RNA-protein crosslinking, to identify transcriptome-wide ∼26,000 HuR binding sites. These sites were on average highly conserved, enriched for HuR binding motifs and mainly located in 3' untranslated regions. Surprisingly, many sites were intronic, implicating HuR in mRNA processing. Upon HuR knockdown, mRNA levels and protein synthesis of thousands of target genes were downregulated, validating functionality. HuR and miRNA binding sites tended to reside nearby but generally did not overlap. Additionally, HuR knockdown triggered strong and specific upregulation of miR-7. In summary, we identified thousands of direct and functional HuR targets, found a human miRNA controlled by HuR, and propose a role for HuR in splicing.

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jekluk
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A systematic RNAi synthetic interaction screen reveals a link between p53 and snoRNP assembly.

Sun, 06/05/2011 - 00:00
Krastev DB, Slabicki M, Paszkowski-Rogacz M, Hubner NC, Junqueira M, Shevchenko A, Mann M, Neugebauer KM, Buchholz F.
Nature Cell Biology
James Blau
Time
12:00pm
TP53(tumour protein 53) is one of the most frequently mutated genes in human cancer and its role during cellular transformation has been studied extensively. However, the homeostatic functions of p53 are less well understood. Here, we explore the molecular dependency network of TP53 through an RNAi-mediated synthetic interaction screen employing two HCT116 isogenic cell lines and a genome-scale endoribonuclease-prepared short interfering RNA library. We identify a variety of TP53 synthetic interactions unmasking the complex connections of p53 to cellular physiology and growth control. Molecular dissection of the TP53 synthetic interaction with UNRIP indicates an enhanced dependency of TP53-negative cells on small nucleolar ribonucleoprotein (snoRNP) assembly. This dependency is mediated by the snoRNP chaperone gene NOLC1 (also known as NOPP140), which we identify as a physiological p53 target gene. This unanticipated function of TP53 in snoRNP assembly highlights the potential of RNAi-mediated synthetic interaction screens to dissect molecular pathways of tumour suppressor genes.
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jblau
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Small regulatory RNAs inhibit RNA polymerase II during the elongation phase of transcription.

Thu, 06/24/2010 - 00:00
Guang S, Bochner AF, Burkhart KB, Burton N, Pavelec DM, Kennedy S.
Nature
Melanie Adams
Time
12:00pm
Eukaryotic cells express a wide variety of endogenous small regulatory RNAs that regulate heterochromatin formation, developmental timing, defence against parasitic nucleic acids and genome rearrangement. Many small regulatory RNAs are thought to function in nuclei1,2. For instance, in plants and fungi, short interfering RNA (siRNAs) associate with nascent transcripts and direct chromatin and/or DNA modifications1,2. To understand further the biological roles of small regulatory RNAs, we conducted a genetic screen to identify factors required for RNA interference (RNAi) in Caenorhabditis elegans nuclei3 . Here we show that the gene nuclear RNAi defective-2 (nrde-2) encodes an evolutionarily conserved protein that is required for siRNA-mediated silencing in nuclei. NRDE-2 associates with the Argonaute protein NRDE-3 within nuclei and is recruited by NRDE-3/siRNA complexes to nascent transcripts that have been targeted by RNAi. We find that nuclear-localized siRNAs direct an NRDE-2-dependent silencing of pre-messenger RNAs (pre-mRNAs) 39 to sites of RNAi, an NRDE-2-dependent accumulation of RNA polymerase (RNAP) II at genomicloci targeted by RNAi, and NRDE-2-dependent decreases in RNAP II occupancy and RNAP II transcriptional activity 39 to sites of RNAi. These results define NRDE-2 as a component of the nuclear RNAi machinery and demonstrate that metazoan siRNAs can silence nuclear-localized RNAs co-transcriptionally. In addition, these results establish a novel mode of RNAP II regulation: siRNA-directed recruitment of NRDE factors that inhibit RNAP II during the elongation phase of transcription.
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Melanie Adams
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