scholarly journals A Genome-wide CRISPR-Cas9 Screen Identifies Host Factors Essential for Optimal Plasmodium Liver Stage Development

2020 ◽  
Author(s):  
Kamalakannan Vijayan ◽  
Nadia Arang ◽  
Ling Wei ◽  
Robert Morrison ◽  
Rechel Geiger ◽  
...  
Keyword(s):  
Microtubule Network ◽  
Liver Stage ◽  
Cytoskeleton Organization ◽  
Guide Rnas ◽  
Genome Wide ◽  
A Genome ◽  
Survival And Development ◽  
Stage Development ◽  
Stage Parasite ◽  
Liver Infection

SummaryPrior to initiating symptomatic malaria, Plasmodium parasites infect and develop within hepatocytes. We performed a forward genetic, genome-wide CRISPR-Cas9 screen to identify host regulators of Plasmodium liver infection. Single guide RNAs targeting genes involved in vesicle trafficking, cytoskeleton organization and lipid biogenesis altered Plasmodium liver development. We observed a redistribution of Golgi-derived vesicles and fragmented Golgi stacks with the parasitophorous vacuolar membrane (PVM). The host microtubule network and non-centrosomal microtubule organizing centers (ncMTOC) also re-localized following infection, closely associating with the parasite. Knocking out the centrosomal MTOC protein CENPJ exasperated the re-localization of MTOCs to the parasite and increased infection, suggesting that the parasite relies on ncMTOC assembly. Thus, we have uncovered a mechanism by which parasites sequester host material for survival and development. Our data provide a wealth of yet untested hypotheses about the elusive biology of the liver stage parasite and serves as a foundation for future investigation.

scholarly journals Optimized strategy for in vivo Cas9-activation in Drosophila

2017 ◽  
Vol 114 (35) ◽  
pp. 9409-9414 ◽  
Author(s):  
Ben Ewen-Campen ◽  
Donghui Yang-Zhou ◽  
Vitória R. Fernandes ◽  
Delfina P. González ◽  
Lu-Ping Liu ◽  
...  
Keyword(s):  
Large Scale ◽  
Target Gene ◽  
In Vivo Studies ◽  
Transcriptional Activators ◽  
Loss Of Function ◽  
Guide Rnas ◽  
Genome Wide ◽  
A Genome ◽  
Endogenous Loci

While several large-scale resources are available for in vivo loss-of-function studies in Drosophila, an analogous resource for overexpressing genes from their endogenous loci does not exist. We describe a strategy for generating such a resource using Cas9 transcriptional activators (CRISPRa). First, we compare a panel of CRISPRa approaches and demonstrate that, for in vivo studies, dCas9-VPR is the most optimal activator. Next, we demonstrate that this approach is scalable and has a high success rate, as >75% of the lines tested activate their target gene. We show that CRISPRa leads to physiologically relevant levels of target gene expression capable of generating strong gain-of-function (GOF) phenotypes in multiple tissues and thus serves as a useful platform for genetic screening. Based on the success of this CRISRPa approach, we are generating a genome-wide collection of flies expressing single-guide RNAs (sgRNAs) for CRISPRa. We also present a collection of more than 30 Gal4 > UAS:dCas9-VPR lines to aid in using these sgRNA lines for GOF studies in vivo.

scholarly journals A genome-wide screen identifies conserved protein hubs required for cadherin-mediated cell–cell adhesion

2014 ◽  
Vol 204 (2) ◽  
pp. 265-279 ◽  
Author(s):  
Christopher P. Toret ◽  
Michael V. D’Ambrosio ◽  
Ronald D. Vale ◽  
Michael A. Simon ◽  
W. James Nelson
Keyword(s):  
Cell Adhesion ◽  
Protein Interactions ◽  
Intracellular Signaling ◽  
Cell Types ◽  
Cytoskeleton Organization ◽  
Drosophila Melanogaster S2 Cells ◽  
Genome Wide ◽  
A Genome ◽  
Protein Functions ◽  
Cell Cell

Cadherins and associated catenins provide an important structural interface between neighboring cells, the actin cytoskeleton, and intracellular signaling pathways in a variety of cell types throughout the Metazoa. However, the full inventory of the proteins and pathways required for cadherin-mediated adhesion has not been established. To this end, we completed a genome-wide (∼14,000 genes) ribonucleic acid interference (RNAi) screen that targeted Ca2+-dependent adhesion in DE-cadherin–expressing Drosophila melanogaster S2 cells in suspension culture. This novel screen eliminated Ca2+-independent cell–cell adhesion, integrin-based adhesion, cell spreading, and cell migration. We identified 17 interconnected regulatory hubs, based on protein functions and protein–protein interactions that regulate the levels of the core cadherin–catenin complex and coordinate cadherin-mediated cell–cell adhesion. Representative proteins from these hubs were analyzed further in Drosophila oogenesis, using targeted germline RNAi, and adhesion was analyzed in Madin–Darby canine kidney mammalian epithelial cell–cell adhesion. These experiments reveal roles for a diversity of cellular pathways that are required for cadherin function in Metazoa, including cytoskeleton organization, cell–substrate interactions, and nuclear and cytoplasmic signaling.

scholarly journals Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells

Science ◽  
2013 ◽  
Vol 343 (6166) ◽  
pp. 84-87 ◽  
Author(s):  
Ophir Shalem ◽  
Neville E. Sanjana ◽  
Ella Hartenian ◽  
Xi Shi ◽  
David A. Scott ◽  
...  
Keyword(s):  
Pluripotent Stem Cells ◽  
Human Cells ◽  
High Rate ◽  
Guide Rnas ◽  
Genome Wide ◽  
A Genome ◽  
Melanoma Model ◽  
Wide Scale ◽  
High Level ◽  
Genome Scale

The simplicity of programming the CRISPR (clustered regularly interspaced short palindromic repeats)–associated nuclease Cas9 to modify specific genomic loci suggests a new way to interrogate gene function on a genome-wide scale. We show that lentiviral delivery of a genome-scale CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequences enables both negative and positive selection screening in human cells. First, we used the GeCKO library to identify genes essential for cell viability in cancer and pluripotent stem cells. Next, in a melanoma model, we screened for genes whose loss is involved in resistance to vemurafenib, a therapeutic RAF inhibitor. Our highest-ranking candidates include previously validated genes NF1 and MED12, as well as novel hits NF2, CUL3, TADA2B, and TADA1. We observe a high level of consistency between independent guide RNAs targeting the same gene and a high rate of hit confirmation, demonstrating the promise of genome-scale screening with Cas9.

scholarly journals Genome-Wide Analysis of C/D and H/ACA-Like Small Nucleolar RNAs in Leishmania major Indicates Conservation among Trypanosomatids in the Repertoire and in Their rRNA Targets

2006 ◽  
Vol 6 (3) ◽  
pp. 361-377 ◽  
Author(s):  
Xue-hai Liang ◽  
Avraham Hury ◽  
Ehud Hoze ◽  
Shai Uliel ◽  
Inna Myslyuk ◽  
...  
Keyword(s):  
Leishmania Major ◽  
Noncoding Rnas ◽  
Structural Features ◽  
Small Nucleolar Rnas ◽  
Human Pathogen ◽  
Genome Wide Analysis ◽  
Guide Rnas ◽  
Genome Wide ◽  
A Genome ◽  
Nucleolar Rnas

ABSTRACT Small nucleolar RNAs (snoRNAs) are a large group of noncoding RNAs that exist in eukaryotes and archaea and guide modifications such as 2′-O-ribose methylations and pseudouridylation on rRNAs and snRNAs. Recently, we described a genome-wide screening approach with Trypanosoma brucei that revealed over 90 guide RNAs. In this study, we extended this approach to analyze the repertoire of the closely related human pathogen Leishmania major. We describe 23 clusters that encode 62 C/Ds that can potentially guide 79 methylations and 37 H/ACA-like RNAs that can potentially guide 30 pseudouridylation reactions. Like T. brucei, Leishmania also contains many modifications and guide RNAs relative to its genome size. This study describes 10 H/ACAs and 14 C/Ds that were not found in T. brucei. Mapping of 2′-O-methylations in rRNA regions rich in modifications suggests the existence of trypanosomatid-specific modifications conserved in T. brucei and Leishmania. Structural features of C/D snoRNAs, such as copy number, conservation of boxes, K turns, and intragenic and extragenic base pairing, were examined to elucidate the great variation in snoRNA abundance. This study highlights the power of comparative genomics for determining conserved features of noncoding RNAs.

Establishment of a genome-wide RNAi screen; Identification of novel genes involved in clonal maintenance of ALL

2014 ◽  
Vol 226 (03) ◽  
Author(s):  
F Ponthan ◽  
D Pal ◽  
J Vormoor ◽  
O Heidenreich
Keyword(s):  
Rnai Screen ◽  
Novel Genes ◽  
Genome Wide ◽  
A Genome

A genome-wide linkage scan for familial partial lipodystrophy susceptibility genes in a German kindreds

2007 ◽  
Vol 30 (4) ◽  
pp. 86
Author(s):  
M. Lanktree ◽  
J. Robinson ◽  
J. Creider ◽  
H. Cao ◽  
D. Carter ◽  
...  
Keyword(s):  
Subcutaneous Fat ◽  
Visceral Obesity ◽  
Fat Distribution ◽  
Dominant Negative ◽  
Molecular Forms ◽  
Partial Lipodystrophy ◽  
Genome Wide ◽  
A Genome ◽  
Familial Partial Lipodystrophy ◽  
Promoter Mutations

Background: In Dunnigan-type familial partial lipodystrophy (FPLD) patients are born with normal fat distribution, but subcutaneous fat from extremities and gluteal regions are lost during puberty. The abnormal fat distribution leads to the development of metabolic syndrome (MetS), a cluster of phenotypes including hyperglycemia, dyslipidemia, hypertension, and visceral obesity. The study of FPLD as a monogenic model of MetS may uncover genetic risk factors of the common MetS which affects ~30% of adult North Americans. Two molecular forms of FPLD have been identified including FPLD2, resulting from heterozygous mutations in the LMNA gene, and FPLD3, resulting from both heterozygous dominant negative and haploinsufficiency mutations in the PPARG gene. However, many patients with clinically diagnosed FPLD have no mutation in either LMNA or PPARG, suggesting the involvement of additional genes in FPLD etiology. Methods: Here, we report the results of an Affymetrix 10K GeneChip microarray genome-wide linkage analysis study of a German kindred displaying the FPLD phenotype and no known lipodystrophy-causing mutations. Results: The investigation identified three chromosomal loci, namely 1q, 3p, and 9q, with non-parametric logarithm of odds (NPL) scores >2.7. While not meeting the criteria for genome-wide significance, it is interesting to note that the 1q and 3p peaks contain the LMNA and PPARG genes respectively. Conclusions: Three possible conclusions can be drawn from these results: 1) the peaks identified are spurious findings, 2) additional genes physically close to LMNA, PPARG, or within 9q, are involved in FPLD etiology, or 3) alternative disease causing mechanisms not identified by standard exon sequencing approaches, such as promoter mutations, alternative splicing, or epigenetics, are also responsible for FPLD.

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