A genome engineering resource to uncover principles of cellular organization and tissue architecture by lipid signaling

Trivedi, Deepti; Cm, Vinitha; Bisht, Karishma; Janardan, Vishnu; Pandit, Awadhesh; Basak, Bishal; Shwetha, H; Ramesh, Navyashree; Raghu, Padinjat

doi:10.7554/elife.55793

Cited by 16 publications

(19 citation statements)

References 48 publications

(39 reference statements)

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“…The ability to use cell lines to optimize CRISPR/Cas9 mutagenesis in non-hemipteran insects is established. These studies have assessed: the ability of sgRNAs and Cas9 to cut target gene sequences, tissue-specific CRISPR, and optimization of Cas13 cutting ( Meltzer et al, 2019 ; Viswanatha et al, 2019 ; Huynh et al, 2020 ; Trivedi et al, 2020 ).…”

Section: Introductionmentioning

confidence: 99%

Gene Editing and Genetic Control of Hemipteran Pests: Progress, Challenges and Perspectives

Pacheco

Walling

Atkinson

2022

Front. Bioeng. Biotechnol.

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The origin of the order Hemiptera can be traced to the late Permian Period more than 230 MYA, well before the origin of flowering plants 100 MY later in during the Cretaceous period. Hemipteran species consume their liquid diets using a sucking proboscis; for phytophagous hemipterans their mouthparts (stylets) are elegant structures that enable voracious feeding from plant xylem or phloem. This adaptation has resulted in some hemipteran species becoming globally significant pests of agriculture resulting in significant annual crop losses. Due to the reliance on chemical insecticides for the control of insect pests in agricultural settings, many hemipteran pests have evolved resistance to insecticides resulting in an urgent need to develop new, species-specific and environmentally friendly methods of pest control. The rapid advances in CRISPR/Cas9 technologies in model insects such as Drosophila melanogaster, Tribolium castaneum, Bombyx mori, and Aedes aegypti has spurred a new round of innovative genetic control strategies in the Diptera and Lepidoptera and an increased interest in assessing genetic control technologies for the Hemiptera. Genetic control approaches in the Hemiptera have, to date, been largely overlooked due to the problems of introducing genetic material into the germline of these insects. The high frequency of CRISPR-mediated mutagenesis in model insect species suggest that, if the delivery problem for Hemiptera could be solved, then gene editing in the Hemiptera might be quickly achieved. Significant advances in CRISPR/Cas9 editing have been realized in nine species of Hemiptera over the past 4 years. Here we review progress in the Hemiptera and discuss the challenges and opportunities for extending contemporary genetic control strategies into species in this agriculturally important insect orderr.

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Section: Introductionmentioning

confidence: 99%

Gene Editing and Genetic Control of Hemipteran Pests: Progress, Challenges and Perspectives

Pacheco

Walling

Atkinson

2022

Front. Bioeng. Biotechnol.

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“…The Vienna Drosophila Stock Center distributes the Heidelberg CRISPR Fly Design (HD_CFD) library [19]. The Bloomington Drosophila Stock Center distributes the TRiP-KO (as well as TRiP-OE for CRISPR activation) library and the WKO library, as well as a number of smaller collections and individual lines [20][21][22][23]. Fly Stocks at the National Institute of Genetics (https://shigen.nig.…”

Section: Transgenic Sgrna Linesmentioning

confidence: 99%

“…At the time of writing the Bloomington Drosophila Stock Center also distributed 99 sgRNA lines generated by the Rhagu lab targeting genes implicated in the regulation of phosphoinositides [23]. These encode two sgRNAs per gene expressed from a U6: 2 promoter.…”

Section: Other Publicly Available Sgrna Collectionsmentioning

confidence: 99%

Tissue-Specific CRISPR-Cas9 Screening in Drosophila

Port

Boutros

2022

Methods in Molecular Biology

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Over the last century research in Drosophila has resulted in many fundamental contributions to our understanding of the biology of multicellular organisms. Many of these breakthroughs have been based on the identification of novel gene functions in large-scale genetic screens. However, conventional forward-genetic screens have been limited by the random nature of mutagenesis and difficulties in mapping causal mutations, while reverse-genetic RNAi screens suffer from incomplete knockdown of gene expression. Recently developed large-scale CRISPR-Cas9 libraries promise to address these limitations by allowing the induction of targeted mutations in genes with spatial and temporal control. Here, we provide a guide for tissue-specific CRISPR screening in Drosophila, including the characterization of Gal4 UAS-Cas9 lines, selection of sgRNA libraries, and various quality control measures. We also discuss confounding factors that can give rise to false-positive and false-negative results in such experiments and suggest strategies on how to detect and avoid them. Conditional CRISPR screening represents an exciting new approach for functional genomics in vivo and is set to further expand our knowledge of the molecular underpinning of development, homeostasis, and disease.

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“…More recently, the exploitation of the bacterial CRISPR/Cas9 system as well as the ribosomal skipping peptide T2A has expanded the toolkit even further with, for example, Trojan exons or CRIMIC in which the integrated cassettes can easily be exchanged for constructs with various other properties [63,64]. A Cas9::T2A::GFP construct has also been implemented as a positive fluorescence marker in a novel CRISPR approach to identify Cas9 expressing cells in which a knock-out could be expected [65]. Raghu Padinjat's group then proceeded to screen for regulatory genes of the phosphatidylinositol signaling pathway necessary for eye development.…”

Section: Other Genetic Screening Approaches In Drosophila Involving Fpsmentioning

confidence: 99%

Application of Fluorescent Proteins for Functional Dissection of the Drosophila Visual System

Smylla

Wagner

Huber

2021

IJMS

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The Drosophila eye has been used extensively to study numerous aspects of biological systems, for example, spatio-temporal regulation of differentiation, visual signal transduction, protein trafficking and neurodegeneration. Right from the advent of fluorescent proteins (FPs) near the end of the millennium, heterologously expressed fusion proteins comprising FPs have been applied in Drosophila vision research not only for subcellular localization of proteins but also for genetic screens and analysis of photoreceptor function. Here, we summarize applications for FPs used in the Drosophila eye as part of genetic screens, to study rhodopsin expression patterns, subcellular protein localization, membrane protein transport or as genetically encoded biosensors for Ca2+ and phospholipids in vivo. We also discuss recently developed FPs that are suitable for super-resolution or correlative light and electron microscopy (CLEM) approaches. Illustrating the possibilities provided by using FPs in Drosophila photoreceptors may aid research in other sensory or neuronal systems that have not yet been studied as well as the Drosophila eye.

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A genome engineering resource to uncover principles of cellular organization and tissue architecture by lipid signaling

Cited by 16 publications

References 48 publications

Gene Editing and Genetic Control of Hemipteran Pests: Progress, Challenges and Perspectives

Gene Editing and Genetic Control of Hemipteran Pests: Progress, Challenges and Perspectives

Tissue-Specific CRISPR-Cas9 Screening in Drosophila

Application of Fluorescent Proteins for Functional Dissection of the Drosophila Visual System

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