RNA Interference (RNAi) Screening in<i>Drosophila</i>

Heigwer, Florian; Port, Fillip; Boutros, Michael

doi:10.1534/genetics.117.300077

Cited by 106 publications

(86 citation statements)

References 210 publications

(266 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Despite these efforts, limitations of RNAi technologies are numerous and consequently advancements in this field have stagnated in recent years. High false negative rates, particularly in highly expressed genes due to insufficient small RNA expression [10,17,21], high false positive rates due to positional or off-target effects [22][23][24][25] and the difficulty of simultaneously targeting multiple genes, has limited the research capabilities of RNAi. Coexpression of Dicer2 can reduce false negative rates, but also may increase the prevalence of false positives [10,17] and further complicates the method.…”

Section: Introductionmentioning

confidence: 99%

Programmable RNA Targeting using CasRx in Flies

Buchman¹,

Brogan²,

Sun³

et al. 2020

Preprint

View full text Add to dashboard Cite

CRISPR-Cas genome editing technologies have revolutionized the fields of functional genetics and genome engineering, but with the recent discovery and optimization of RNA-targeting Cas ribonucleases, we may soon see a similar revolution in the study of RNA function and transcriptome engineering. However, to date, successful proof-of-principle for Cas ribonuclease RNA targeting in eukaryotic systems has been limited. Only recently has successful modification of RNA expression by a Cas ribonuclease been demonstrated in animal embryos. This previous work, however, did not evaluate endogenous expression of Cas ribonucleases and only focused on Cas ribonuclease function in early developmental stages. A more comprehensive evaluation of this technology is needed to assess its potential impact in the field. Here we report on our efforts to develop a programmable platform for RNA-targeting using a Cas ribonuclease, CasRx, in the model organism Drosophila melanogaster. By genetically encoding CasRx in flies, we demonstrate moderate transcript targeting of known phenotypic genes in addition to unexpected toxicity and lethality. We also report on the off-target effects following on-target transcript cleavage by CasRx. Taken together, our results present the current state and limitations of a genetically encoded programmable RNA-targeting Cas system in Drosophila melanogaster, paving the way for future optimization of the system.

show abstract

Section: Introductionmentioning

confidence: 99%

Programmable RNA Targeting using CasRx in Flies

Buchman¹,

Brogan²,

Sun³

et al. 2020

Preprint

View full text Add to dashboard Cite

show abstract

“…LOF was classically achieved in vivo in Drosophila using random mutagenesis (St Johnston 2002) and in more recent years, is achieved by knockdown using RNA interference (RNAi) (Dietzl et al 2007), or knockout using CRISPR-Cas9 (Port et al 2014). Transgenic RNAi is a powerful and straightforward method for analysis of gene function [reviews include (Perrimon et al 2010;Heigwer et al 2018)]. In Drosophila, RNAi is cell-autonomous, and is used in conjunction with the GAL4/UAS system (Brand and Perrimon 1993) for both spatial and temporal knockdown, providing a powerful approach to the study of genes with pleiotropic functions.…”

Section: Introductionmentioning

confidence: 99%

Large-scale transgenicDrosophilaresource collections for loss- and gain-of-function studies

Zirin

Liu

et al. 2019

Preprint

View full text Add to dashboard Cite

The Transgenic RNAi Project (TRiP), a Drosophila functional genomics platform at Harvard Medical School, was initiated in 2008 to generate and distribute a genome-scale collection of RNAi fly stocks. To date, the TRiP has generated >15,000 RNAi fly stocks. As this covers most Drosophila genes, we have largely transitioned to development of new resources based on CRISPR technology. Here, we present an update on our libraries of publicly available RNAi and CRISPR fly stocks, and focus on the TRiP-CRISPR overexpression (TRiP-OE) and TRiP-CRISPR knockout (TRiP-KO) collections. TRiP-OE stocks express sgRNAs targeting upstream of a gene transcription start site. Gene activation is triggered by co-expression of catalytically dead Cas9 (dCas9) fused to an activator domain, either VP64-p65-Rta (VPR) or Synergistic Activation Mediator (SAM). TRiP-KO stocks express one or two sgRNAs targeting the coding sequence of a gene or genes, allowing for generation of indels in both germline and somatic tissue. To date, we have generated more than 5,000 CRISPR-OE or -KO stocks for the community. These resources provide versatile, transformative tools for gene activation, gene repression, and genome engineering.

show abstract

“…In contrast, reverse genetic approaches, such as RNA interference (RNAi), are gene-centric designed and allow to probe the function of a large number of genes (Boutros and Ahringer, 2008;Mohr et al, 2014;Heigwer et al, 2018;Horn et al, 2011). Furthermore, RNAi reagents can be genetically encoded and used to screen for gene function with spatial and temporal precision (Dietzl et al, 2007;Kaya-Çopur and Schnorrer, 2016;Ni et al, 2009).…”

Section: Introductionmentioning

confidence: 99%

“…Furthermore, RNAi reagents can be genetically encoded and used to screen for gene function with spatial and temporal precision (Dietzl et al, 2007;Kaya-Çopur and Schnorrer, 2016;Ni et al, 2009). In recent years tissue specific-RNAi screens have provided multiple new insights into the biology of multicellular animals (reviewed in Heigwer et al, 2018). However, RNAi is often limited by incomplete penetrance due to residual gene expression and can suffer from off-target effects (Perkins et al, 2015;Echeverri et al, 2006;Ma et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

A large-scale resource for tissue-specific CRISPR mutagenesis inDrosophila

Port

Strein

Stricker

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

Genetic screens are powerful tools for the functional annotation of genomes. In the context of multicellular organisms, interrogation of gene function is greatly facilitated by methods that allow spatial and temporal control of gene abrogation. Here, we describe a large-scale transgenic short guide (sg) RNA library for efficient CRISPR-based disruption of specific target genes in a constitutive or conditional manner. The library consists currently of more than 2600 plasmids and 1400 fly lines with a focus on targeting kinases, phosphatases and transcription factors, each expressing two sgRNAs under control of the Gal4/UAS system. We show that conditional CRISPR mutagenesis is robust across many target genes and can be efficiently employed in various somatic tissues, as well as the germline. In order to prevent artefacts commonly associated with excessive amounts of Cas9 protein, we have developed a series of novel UAS-Cas9 transgenes, which allow fine tuning of Cas9 expression to achieve high gene editing activity without detectable toxicity. Functional assays, as well as direct sequencing of genomic sgRNA target sites, indicates that the vast majority of transgenic sgRNA lines mediate efficient gene disruption. Furthermore, we conducted the so far largest fully transgenic CRISPR screen in any metazoan organism, which further supported the high efficiency and accuracy of our library and revealed many so far uncharacterized genes essential for development.

show abstract

RNA Interference (RNAi) Screening inDrosophila

Cited by 106 publications

References 210 publications

Programmable RNA Targeting using CasRx in Flies

Programmable RNA Targeting using CasRx in Flies

Large-scale transgenicDrosophilaresource collections for loss- and gain-of-function studies

A large-scale resource for tissue-specific CRISPR mutagenesis inDrosophila

Contact Info

Product

Resources

About