Author Correction: A one-step tRNA-CRISPR system for genome-wide genetic interaction mapping in mammalian cells

Abstract Mapping genetic interactions in mammalian cells is limited due to technical obstacles. Here we describe a method called TCGI (tRNA-CRISPR for genetic interactions) to generate a high-efficient, barcode-free and scalable pairwise CRISPR libraries in mammalian cells for identifying genetic interactions. We have generated a genome- wide library to identify genes genetically interacting with TAZ in cell viability regulation. Validation of candidate synergistic genes reveals the screening accuracy of 85% and TAZ-MCL1 is characterized as combinational drug targets for non-small cell lung cancer treatments. TCGI has dramatically improved the current methods for mapping genetic interactions and screening drug targets for combinational therapies.

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Genetic screens in isogenic mammalian cell lines without single cell cloning

10.1101/677385 ◽

2019 ◽

Cited By ~ 1

Author(s):

Peter C DeWeirdt ◽

Kendall R Sanson ◽

Ruth E Hanna ◽

Mudra Hegde ◽

Annabel K Sangree ◽

...

Keyword(s):

Cell Lines ◽

Mammalian Cells ◽

Genetic Interaction ◽

Cell Types ◽

Repair Gene ◽

Synthetic Lethal ◽

Mammalian Cell Lines ◽

Genome Wide ◽

Single Cell Cloning ◽

Synthetic Lethal Interactions

Isogenic pairs of cell lines, which differ by a single genetic modification, are powerful tools for understanding gene function. Generating such pairs for mammalian cells, however, is labor-intensive, time-consuming, and impossible in some cell types. Here we present an approach to create isogenic pairs of cells and screen them with genome-wide CRISPR-Cas9 libraries to generate genetic interaction maps. We queried the anti-apoptotic genes BCL2L1 and MCL1, and the DNA damage repair gene PARP1, via 25 genome-wide screens across 4 cell lines. For all three genes, we identify a rich set of both expected and novel buffering and synthetic lethal interactions. Further, we compare the interactions observed in genetic space to those found when targeting these genes with small molecules and identify hits that may inform the clinical uses for these inhibitors. We anticipate that this methodology will be broadly useful to comprehensively study genes of interest across many cell types.

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The cellular response to drug perturbation is limited: comparison of large-scale chemogenomic fitness signatures

10.21203/rs.3.rs-781592/v1 ◽

2021 ◽

Author(s):

Marjan Barazandeh ◽

Divya Kriti ◽

Corey Nislow ◽

Guri Giaever

Keyword(s):

Small Molecules ◽

Mammalian Cells ◽

Cellular Response ◽

Large Scale ◽

Genetic Interaction ◽

Common Mechanism ◽

Genome Wide ◽

Powerful Approach ◽

Response Systems ◽

Academic Laboratory

Abstract BackgroundChemogenomic profiling is a powerful approach towards understanding the genome-wide cellular response to small molecules. Developed in Saccharomyces cerevisiae, chemogenomic screens provide direct, unbiased identification of drug target candidates as well as genes required for drug resistance. While many laboratories have performed chemogenomic fitness assays, they have not been assessed for reproducibility and accuracy. Here we analyze the two largest independent yeast chemogenomic datasets comprising over 35 million gene-drug interactions and more than 6000 unique chemogenomic profiles; the first from our own academic laboratory and the second from the Novartis Institute of Biomedical Research (NIBR).ResultsCombining the datasets revealed robust genetic interaction response signatures that point to common mechanism of action, despite the substantial differences in experimental and analytical pipelines. We previously reported that the cellular response to small molecules is limited and can be described by a network of 45 chemogenomic signatures. In the present study, we show that the majority of these signatures (66%) are also found in the companion dataset, providing further support for their biological relevance as systems-level, small molecule response systems. ConclusionsOur results demonstrate the robustness of chemogenomic fitness profiling in yeast, while offering guidelines for performing other high-dimensional comparisons including parallel CRISPR screens in mammalian cells.

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Integrated platform for genome-wide screening and construction of high-density genetic interaction maps in mammalian cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1307002110 ◽

2013 ◽

Vol 110 (25) ◽

pp. E2317-E2326 ◽

Cited By ~ 81

Author(s):

M. Kampmann ◽

M. C. Bassik ◽

J. S. Weissman

Keyword(s):

Mammalian Cells ◽

Genetic Interaction ◽

High Density ◽

Genome Wide ◽

Integrated Platform

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Genome-wide mapping of unexplored essential regions in the Saccharomyces cerevisiae genome: evidence for hidden synthetic lethal combinations in a genetic interaction network

Nucleic Acids Research ◽

10.1093/nar/gku576 ◽

2014 ◽

Vol 42 (15) ◽

pp. 9838-9853 ◽

Cited By ~ 6

Author(s):

Saeed Kaboli ◽

Takuya Yamakawa ◽

Keisuke Sunada ◽

Tao Takagaki ◽

Yu Sasano ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Genetic Interaction ◽

Interaction Network ◽

Essential Genes ◽

Genetic Interactions ◽

Lethal Gene ◽

Synthetic Lethal ◽

Genome Wide ◽

A Genome ◽

Wide Scale

Abstract Despite systematic approaches to mapping networks of genetic interactions in Saccharomyces cerevisiae, exploration of genetic interactions on a genome-wide scale has been limited. The S. cerevisiae haploid genome has 110 regions that are longer than 10 kb but harbor only non-essential genes. Here, we attempted to delete these regions by PCR-mediated chromosomal deletion technology (PCD), which enables chromosomal segments to be deleted by a one-step transformation. Thirty-three of the 110 regions could be deleted, but the remaining 77 regions could not. To determine whether the 77 undeletable regions are essential, we successfully converted 67 of them to mini-chromosomes marked with URA3 using PCR-mediated chromosome splitting technology and conducted a mitotic loss assay of the mini-chromosomes. Fifty-six of the 67 regions were found to be essential for cell growth, and 49 of these carried co-lethal gene pair(s) that were not previously been detected by synthetic genetic array analysis. This result implies that regions harboring only non-essential genes contain unidentified synthetic lethal combinations at an unexpectedly high frequency, revealing a novel landscape of genetic interactions in the S. cerevisiae genome. Furthermore, this study indicates that segmental deletion might be exploited for not only revealing genome function but also breeding stress-tolerant strains.

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A Novel Type V CRISPR System with Potential for Genome Editing in the Liver

Blood ◽

10.1182/blood-2021-154505 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1862-1862

Author(s):

Gregory J. Cost ◽

Morayma Temoche-Diaz ◽

Janet Mei ◽

Cristina N. Butterfield ◽

Christopher T. Brown ◽

...

Keyword(s):

Amino Acid ◽

Genome Editing ◽

Mammalian Cells ◽

Conflicts Of Interest ◽

Attractive Alternative ◽

Vitro Assay ◽

Crispr System ◽

Type V

Abstract RNA guided CRISPR genome editing systems can make specific changes to the genomes of mammalian cells and have the potential to treat a range of diseases including those that can be addressed by editing hepatocytes. Attempts to edit the liver in vivo have relied almost exclusively on the Cas9 nucleases derived from the bacteria S treptococcus pyogenes or Staphylococcus aureus to which humans are commonly exposed. Pre-existing immunity to both these proteins has been reported in humans which raises concerns about their in vivo application. In silico analysis of a large metagenomics database followed by testing in mammalian cells in culture identified MG29-1, a novel CRISPR system which is a member of the Type V family but exhibits only 41 % amino acid identity to Francisella tularensis Cas12a/cpf1. MG29-1 is a 1280 amino acid RNA programmable nuclease that utilizes a single guide RNA comprised of a 22 nucleotide (nt) constant region and a 20 to 25 nt spacer, recognizes the PAM KTTN (predicted frequency 1 in 16 bp) and generates staggered cuts. MG29-1 was derived from a sample taken from a hydrothermal vent and it is therefore unlikely that humans will have developed pre-existing immunity to this protein. A screen for sgRNA targeting serum albumin in the mouse liver cell line Hepa1-6 identified 6 guides that generated more than 80% INDELS. The MG29-1 system was optimized for in vivo delivery by screening chemical modifications to the guide that improve stability in mammalian cell lysates while retaining or improving editing activity. Two lead guide chemistries were evaluated in mice using MG29-1 mRNA and sgRNA packaged in lipid nanoparticles (LNP). Three days after a single IV administration on-target editing was evaluated in the liver by Sanger sequencing. The sgRNA that was the most stable in the in vitro assay generated INDELS that ranged from 20 to 25% while a sgRNA with lower in vitro stability failed to generate detectable INDELs. The short sgRNA and small protein size compared to spCas9 makes MG29-1 an attractive alternative to spCas9 for in vivo editing applications. Evaluation of the potential of MG29-1 to perform gene knockouts and gene additions via non-homologous end joining is ongoing. Disclosures No relevant conflicts of interest to declare.

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Controlling quality and amount of mitochondria by mitophagy: insights into the role of ubiquitination and deubiquitination

Biological Chemistry ◽

10.1515/hsz-2016-0125 ◽

2016 ◽

Vol 397 (7) ◽

pp. 637-647 ◽

Cited By ~ 12

Author(s):

Tao Tan ◽

Marcel Zimmermann ◽

Andreas S. Reichert

Keyword(s):

Mammalian Cells ◽

Mitochondrial Fission ◽

Baker’S Yeast ◽

Negative Regulator ◽

Mitochondrial Outer Membrane ◽

Baker's Yeast ◽

Genome Wide ◽

A Genome ◽

Autophagy Pathway ◽

Quantity Control

Abstract Mitophagy is a selective autophagy pathway conserved in eukaryotes and plays an essential role in mitochondrial quality and quantity control. Mitochondrial fission and fusion cycles maintain a certain amount of healthy mitochondria and allow the isolation of damaged mitochondria for their elimination by mitophagy. Mitophagy can be classified into receptor-dependent and ubiquitin-dependent pathways. The mitochondrial outer membrane protein Atg32 is identified as the only known receptor for mitophagy in baker’s yeast, whereas mitochondrial proteins FUNDC1, NIX/BNIP3L, BNIP3 and Bcl2L13 are recognized as mitophagy receptors in mammalian cells. Earlier studies showed that ubiquitination and deubiquitination occurs in yeast, yet there is no direct evidence for an ubiquitin-dependent mitophagy pathway in this organism. In contrast, a ubiquitin-/PINK1-/Parkin-dependent mitophagy pathway was unraveled and was extensively characterized in mammals in recent years. Recently, a quantitative method termed synthetic quantitative array (SQA) technology was developed to identify modulators of mitophagy in baker’s yeast on a genome-wide level. The Ubp3-Bre5 deubiquitination complex was found as a negative regulator of mitophagy while promoting other autophagic pathways. Here we discuss how ubiquitination and deubiquitination regulates mitophagy and other selective forms of autophagy and what argues for using baker’s yeast as a model to study the ubiquitin-dependent mitophagy pathway.

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