An optimized TALEN application for mutagenesis and screening in<i>Drosophila melanogaster</i>

Lee, Han B.; Sebo, Zachary L; Peng, Ying; Guo, Yi

doi:10.1080/21592799.2015.1023423

Cited by 11 publications

(4 citation statements)

References 63 publications

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“…Designing TALEsissimpler than designing zinc-finger-containing proteins,howeverusing the TALE systemto create specific TALE-endonucleases (TALENs) still requires extensive gene-construction orsynthesis. There have been a number of reports, mostly technical in nature, COIS 2015 DRAFT 4 demonstrating the functionality of TALENs in various insect species and the conditions under which they function best [15,20,23,[25][26][27][28][29][30][31][32][33][34][35][36].…”

Section: Cois 2015 Draftmentioning

confidence: 99%

“…Cas9 is a DNA endonuclease associated with the CRISPR/Cas system found in Streptococcus pyogenes.The DNA sequence specificity of Cas9 is determinedby by small associated RNAs (crRNA and tracrRNA; in the lab these are combined to form a single RNA referred to as a guideRNA or gRNA) [37]. Unlike ZFNs and TALENs, Cas9 gene editing does not requirerepeatedly designing and expressing new Cas9 proteins, but instead onlyrequires producing short target-specific gRNAs that associate with Cas9to confer the desired site specificity [38].Despite only recently becoming available as a gene editing system, Cas9 is quickly being adopted by insect biologists although most uses in insects to date have been technical studies evaluating the performance characteristics of the system [20,32,35,36,].…”

Section: Crispr/casmentioning

confidence: 99%

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Applications of genome editing in insects

Reid

O’Brochta

2016

Current Opinion in Insect Science

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Insect genome editing was first reported 1991 in Drosophila melanogaster but the technology used was not portable to other species. Not until the recent development of facile, engineered DNA endonuclease systems has gene editing become widely available to insect scientists. Most applications in insects to date have been technical in nature but this is rapidly changing. Functional genomics and genetics-based insect control efforts will be major beneficiaries of the application of contemporary gene editing technologies. Engineered endonucleases like Cas9 make it possible to create powerful and effective gene drive systems that could be used to reduce or even eradicate specific insect populations. 'Best practices' for using Cas9-based editing are beginning to emerge making it easier and more effective to design and use but gene editing technologies still require traditional means of delivery in order to introduce them into somatic and germ cells of insects-microinjection of developing embryos. This constrains the use of these technologies by insect scientists. Insects created using editing technologies challenge existing governmental regulatory structures designed to manage genetically modified organisms.

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Section: Cois 2015 Draftmentioning

confidence: 99%

Section: Crispr/casmentioning

confidence: 99%

Applications of genome editing in insects

Reid

O’Brochta

2016

Current Opinion in Insect Science

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“…To validate the efficiency of gRNA-Ptp69D , we established a “Cas9-LEThAL” (for Cas9- induced lethal e ffect th rough the a bsence of L ig4) assay (Fig. S2) that was adapted from a previously described method for assessing injection-based gRNA efficiency 38 . Efficient gRNAs for non-essential genes, such as a published gRNA for e 18 (Table S2), cause male-specific lethality in pupal stages when males carrying gRNAs are crossed to Act-Cas9 lig4 homozygous females.…”

Section: Resultsmentioning

confidence: 99%

Highly efficient CRISPR/Cas9-mediated tissue specific mutagenesis inDrosophila

Wang

et al. 2018

Preprint

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Tissue-specific loss-of-function (LOF) analysis is an essential approach for characterizing gene function. Here we describe an efficient CRISPR-mediated tissue-restricted mutagenesis (CRISPR-TRiM) method for ablating gene function in Drosophila. This binary system consists of a tissue-specific Cas9 and a ubiquitously expressed multi-guide RNA (gRNA) transgene. To facilitate the construction of these components, we developed convenient tools for generating and evaluating enhancer-driven Cas9 lines, identified a multi-gRNA design that is highly efficient in mutagenizing somatic cells, and established an assay for testing the efficiency of multi-gRNAs in creating double-stranded breaks. We found that excision of genomic DNA induced by two gRNAs is infrequent in somatic cells, while indels more reliably cause tissue-specific LOF. Furthermore, we show that enhancer-driven Cas9 is less cytotoxic yet results in more complete gene removal than Gal4-driven Cas9 in larval neurons. Finally, we demonstrate that CRISPR-TRiM efficiently unmasks redundant gene functions in neuronal morphogenesis. Importantly, two Cas9 transgenes that turn on with different timings in the neuronal lineage revealed the extent to which gene products persist in cells after tissue-specific gene knockout. These CRISRPR tools can be applied to analyze tissue-specific gene function in many biological processes.

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“…The assay using the enzyme is used to confirm the presence or absence of mutation introduction in genome-editing experiments (TALE nuclease, CRISPR/Cas9, etc.) (23)(24)(25). In another study, the SN assay was used to screen for mutations in kidney-related genes.…”

mentioning

confidence: 99%

A Simple Method To Detect Point Mutations in Aspergillus fumigatus cyp51A Gene Using a Surveyor Nuclease Assay

Arai

Majima

Watanabe

et al. 2020

Antimicrob Agents Chemother

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One of the main mechanisms of azole resistance of Aspergillus fumigatus is thought to be a reduction in the drug’s affinity for the target molecule, Cyp51A, due to its amino acid mutation(s). It is known that the azole resistance pattern is closely related to the mutation site(s) of the molecule. In this study, we tried to develop a simple and rapid detection method for cyp51A mutations using the endonuclease Surveyor nuclease. The Surveyor nuclease assay was verified using several azole-resistant strains of A. fumigatus that possess point mutations in Cyp51A. For validation of the Surveyor nuclease assay, blind tests were conducted using 48 strains of A. fumigatus (17 azole-resistant and 31 azole-susceptible strains). The Surveyor nuclease assay could rapidly detect cyp51A mutations with one primer set. Also, all the tested strains harboring different cyp51A single point mutations could be clearly distinguished from the wild type. The Surveyor nuclease assay is a simple method that can detect cyp51A mutations rapidly.

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An optimized TALEN application for mutagenesis and screening inDrosophila melanogaster

Cited by 11 publications

References 63 publications

Applications of genome editing in insects

Applications of genome editing in insects

Highly efficient CRISPR/Cas9-mediated tissue specific mutagenesis inDrosophila

A Simple Method To Detect Point Mutations in Aspergillus fumigatus cyp51A Gene Using a Surveyor Nuclease Assay

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