CRISPR/Cas9 Mediates Efficient Conditional Mutagenesis in<i>Drosophila</i>

Xue, Zhaoyu; Wu, Menghua; Wen, Kejia; Ren, Menda; Long, Li; Zhang, Xuedi; Gao, Guanjun

doi:10.1534/g3.114.014159

Cited by 85 publications

(83 citation statements)

References 32 publications

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“…Although CRISPR mutagenesis using stable transgenic lines expressing both cas9 and sgRNA have been used for generating both germline and somatic mutations in nonvertebrate models such as Caenorhabditis elegans and Drosophila melanogaster (Port et al 2014;Shen et al 2014;Xue et al 2014), it has not been reported in vertebrates until our manuscript was being revised (Ablain et al 2015). A Cas9 knock-in mouse line has been used to achieve tissue-specific mutagenesis (Platt et al 2014;Swiech et al 2015).…”

Section: Discussionmentioning

confidence: 99%

Multiplex Conditional Mutagenesis Using Transgenic Expression of Cas9 and sgRNAs

et al. 2015

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Determining the mechanism of gene function is greatly enhanced using conditional mutagenesis. However, generating engineered conditional alleles is inefficient and has only been widely used in mice. Importantly, multiplex conditional mutagenesis requires extensive breeding. Here we demonstrate a system for one-generation multiplex conditional mutagenesis in zebrafish (Danio rerio) using transgenic expression of both cas9 and multiple single guide RNAs (sgRNAs). We describe five distinct zebrafish U6 promoters for sgRNA expression and demonstrate efficient multiplex biallelic inactivation of tyrosinase and insulin receptor a and b, resulting in defects in pigmentation and glucose homeostasis. Furthermore, we demonstrate temporal and tissue-specific mutagenesis using transgenic expression of Cas9. Heat-shock-inducible expression of cas9 allows temporal control of tyr mutagenesis. Liver-specific expression of cas9 disrupts insulin receptor a and b, causing fasting hypoglycemia and postprandial hyperglycemia. We also show that delivery of sgRNAs targeting ascl1a into the eye leads to impaired damage-induced photoreceptor regeneration. Our findings suggest that CRISPR/Cas9-based conditional mutagenesis in zebrafish is not only feasible but rapid and straightforward.KEYWORDS CRISPR/Cas9; conditional mutagenesis; glucose homeostasis; retinal regeneration; zebrafish C ONDITIONAL gene inactivation is necessary for determining physiological functions of genes whose conventional mutation causes embryonic lethality or multiorgan defects. It has been widely used in mice because of the availability of embryonic stem (ES) cells and large collections of both ES cell lines and animals that carry conditional alleles. The conditional alleles usually contain strategically placed Cre or Flp target sites in introns that permit deletion of the intervening exon(s) (Gu et al. 1994). Some conditional alleles harbor a Cre and/or Flp invertible gene trap in an intron that allows an on/off switch of transcription (Schnutgen et al. 2005;Ni et al. 2012). The function or expression of these alleles is "switched" off in the presence of Cre or Flp activity. In Drosophila, conditional gene inactivation can be achieved using RNA interference (RNAi), and genome-wide libraries of RNAi transgenes are available (Dietzl et al. 2007;Ni et al. 2008). For the increasingly popular zebrafish model, however, only limited conditional alleles generated from invertible gene trapping are available (Ni et al. 2012). The lack of ES cells and the inefficiency of RNAi in zebrafish necessitate new approaches for targeted conditional gene inactivation.CRISPR/Cas9 mutagenesis has been applied to many model systems from cultured cells to whole organisms (Doudna and Charpentier 2014;Hsu et al. 2014). The system only requires a two-component RNA-protein complex (RNP): a single guide RNA (sgRNA) that identifies the target through base pairing and the Cas9 endonuclease that generates double-strand breaks (DSBs) at the target site upon sgRNA-target base pairing. CRI...

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Section: Discussionmentioning

confidence: 99%

Multiplex Conditional Mutagenesis Using Transgenic Expression of Cas9 and sgRNAs

et al. 2015

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“…It can easily be adapted for use in species other than the zebrafish by modifying the codon-optimized Cas9 and U6 promoter sequences. Note that systems for tissue-specific gene inactivation have also been developed in fly and worm (Shen et al, 2014; Xue et al, 2014). We have also added a second U6:gRNA cassette in the vector backbone to allow for multiplexing.…”

Section: Discussionmentioning

confidence: 99%

Tissue-specific gene targeting using CRISPR/Cas9

Ablain

Zon

2016

Methods in Cell Biology

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The zebrafish has been a powerful model in forward genetic screens to identify genes essential for organogenesis and embryonic development. Conversely, using reverse genetics to investigate specific gene function requires phenotypic analysis of complete gene inactivation. Despite the availability and efficacy of morpholinos, the lack of tractable and efficient knockout technologies has impeded reverse genetic studies in the zebrafish, particularly in adult animals. The recent development of genome-editing technologies such as CRISPR/Cas9 greatly widened the scope of loss-of-function studies in the zebrafish, allowing for the rapid phenotypic assessment of gene silencing in embryos, the generation of knockout lines, and large-scale reverse genetic screens. Tissue-specific gene inactivation would be ideal for these studies given the caveats of whole-embryo gene silencing, yet spatial control of gene targeting remains a challenge. In this chapter, we focus on tissue-specific gene inactivation using the CRISPR/Cas9 technology. We first explain the rationale for this technique, including some of its potential applications to tackle important biological issues and the inability of current technologies to address these issues. We then present a method to target genes in a tissue-specific manner in the zebrafish. Finally, we discuss technical difficulties and limitations of this method as well as possible future developments.

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“…By using different patterns of Gal4 expression, tissue-or stage-specific gene editing can be realized. These strategies have proved feasible in B. mori and Drosophila [84][85][86].…”

Section: Challengesmentioning

confidence: 99%

Advanced technologies for genetically manipulating the silkwormBombyx mori, a model Lepidopteran insect

O’Brochta

2015

Proc. R. Soc. B.

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Genetic technologies based on transposon-mediated transgenesis along with several recently developed genome-editing technologies have become the preferred methods of choice for genetically manipulating many organisms. The silkworm, Bombyx mori, is a Lepidopteran insect of great economic importance because of its use in silk production and because it is a valuable model insect that has greatly enhanced our understanding of the biology of insects, including many agricultural pests. In the past 10 years, great advances have been achieved in the development of genetic technologies in B. mori, including transposon-based technologies that rely on piggyBac-mediated transgenesis and genome-editing technologies that rely on protein-or RNA-guided modification of chromosomes. The successful development and application of these technologies has not only facilitated a better understanding of B. mori and its use as a silk production system, but also provided valuable experiences that have contributed to the development of similar technologies in non-model insects. This review summarizes the technologies currently available for use in B. mori, their application to the study of gene function and their use in genetically modifying B. mori for biotechnology applications. The challenges, solutions and future prospects associated with the development and application of genetic technologies in B. mori are also discussed.

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CRISPR/Cas9 Mediates Efficient Conditional Mutagenesis inDrosophila

Cited by 85 publications

References 32 publications

Multiplex Conditional Mutagenesis Using Transgenic Expression of Cas9 and sgRNAs

Multiplex Conditional Mutagenesis Using Transgenic Expression of Cas9 and sgRNAs

Tissue-specific gene targeting using CRISPR/Cas9

Advanced technologies for genetically manipulating the silkwormBombyx mori, a model Lepidopteran insect

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