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CRISPR/Cas9 mediates efficient conditional mutagenesis in Drosophila.

Xue Z, Wu M, Wen K, Ren M, Long L, Zhang X, Gao G - G3 (Bethesda) (2014)

Bottom Line: Existing transgenic RNA interference (RNAi) methods greatly facilitate functional genome studies via controlled silencing of targeted mRNA in Drosophila.Although the RNAi approach is extremely powerful, concerns still linger about its low efficiency.Here, we developed a CRISPR/Cas9-mediated conditional mutagenesis system by combining tissue-specific expression of Cas9 driven by the Gal4/upstream activating site system with various ubiquitously expressed guide RNA transgenes to effectively inactivate gene expression in a temporally and spatially controlled manner.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Tsinghua University, Beijing 100084, China.

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A schematic overview of the CMCM system. For each gene of interest (gene X), transgenic stocks of U6B-geneX-gRNA, which ubiquitously express gRNA targeting the gene, were established. Transgenic 10ΧUAS-Cas9/U6B-geneX-gRNA male flies were recovered after crossing the 10XUAS-Cas9 stock with the U6B-geneX-gRNA stock. The recovered male flies were crossed with the enhancer-trap Gal4 virgin flies to drive Cas9 expression in specific tissues. Mutations were induced only in the specific tissue determined by the Gal4 stocks.
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fig1: A schematic overview of the CMCM system. For each gene of interest (gene X), transgenic stocks of U6B-geneX-gRNA, which ubiquitously express gRNA targeting the gene, were established. Transgenic 10ΧUAS-Cas9/U6B-geneX-gRNA male flies were recovered after crossing the 10XUAS-Cas9 stock with the U6B-geneX-gRNA stock. The recovered male flies were crossed with the enhancer-trap Gal4 virgin flies to drive Cas9 expression in specific tissues. Mutations were induced only in the specific tissue determined by the Gal4 stocks.

Mentions: Recently, the RNA-guided CRISPR/Cas9 technology has shown potential for highly efficient genome editing in many organisms, including Drosophila (Bassett et al. 2013; Golic 2013; Gratz et al. 2013, 2014; Yu et al. 2013). Stable expression of Cas9 nuclease driven by a germline-specific promoter can induce efficient germline-transmitted mutagenesis in F1 progeny (Kondo and Ueda 2013; Ren et al. 2013; Xue et al. 2014). In addition, the CRISPR/Cas9 system has been successfully applied for conditional genome editing in C. elegans, mouse, and rat (Ma et al. 2013; Yang et al. 2013; Liu et al. 2014). More recently, UAS-driven expression of Cas9 in Drosophila has been shown to work with the CRISPR/Cas9 system (Port et al. 2014). Based on the high efficiency of Cas9/gRNA gene targeting, we reasoned that directly disrupting targeted genes at the DNA level would destroy gene function more efficiently than posttranscriptional breakdown of the targeted mRNA mediated by RNAi. Therefore, we combined the CRISPR/Cas9 and Gal4-UAS systems to develop a CRISPR/Cas9-mediated conditional mutagenesis (CMCM) method and systematically investigated whether it can efficiently inactivate gene activity in most Drosophila tissues. This method contains two steps (Figure 1). First, we generated gene-specific gRNAs directed against a number of genes with known phenotypes and made their 10UAS-Cas9/gRNAs transgenic flies. Second, we induced the expression of 10UAS-Cas9/gRNAs driven by the tissue-specific Gal4 lines and investigated the efficiency of gene disruption and associated phenotypes. Using the CMCM system, we tested six genes (y, notch, bam, nos, cid, and ms(3)k81) and achieved highly effective disruption in wing, eye, ovary, and testis tissues. Moreover, side-by-side comparisons of tissue-specific gene disruption showed the power of the CMCM system. In general, it was more efficient and rapid than RNAi approaches and provided a novel genetic tool for studying gene function in specific tissues in Drosophila (Dietzl et al. 2007; Ni et al. 2011).


CRISPR/Cas9 mediates efficient conditional mutagenesis in Drosophila.

Xue Z, Wu M, Wen K, Ren M, Long L, Zhang X, Gao G - G3 (Bethesda) (2014)

A schematic overview of the CMCM system. For each gene of interest (gene X), transgenic stocks of U6B-geneX-gRNA, which ubiquitously express gRNA targeting the gene, were established. Transgenic 10ΧUAS-Cas9/U6B-geneX-gRNA male flies were recovered after crossing the 10XUAS-Cas9 stock with the U6B-geneX-gRNA stock. The recovered male flies were crossed with the enhancer-trap Gal4 virgin flies to drive Cas9 expression in specific tissues. Mutations were induced only in the specific tissue determined by the Gal4 stocks.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4232542&req=5

fig1: A schematic overview of the CMCM system. For each gene of interest (gene X), transgenic stocks of U6B-geneX-gRNA, which ubiquitously express gRNA targeting the gene, were established. Transgenic 10ΧUAS-Cas9/U6B-geneX-gRNA male flies were recovered after crossing the 10XUAS-Cas9 stock with the U6B-geneX-gRNA stock. The recovered male flies were crossed with the enhancer-trap Gal4 virgin flies to drive Cas9 expression in specific tissues. Mutations were induced only in the specific tissue determined by the Gal4 stocks.
Mentions: Recently, the RNA-guided CRISPR/Cas9 technology has shown potential for highly efficient genome editing in many organisms, including Drosophila (Bassett et al. 2013; Golic 2013; Gratz et al. 2013, 2014; Yu et al. 2013). Stable expression of Cas9 nuclease driven by a germline-specific promoter can induce efficient germline-transmitted mutagenesis in F1 progeny (Kondo and Ueda 2013; Ren et al. 2013; Xue et al. 2014). In addition, the CRISPR/Cas9 system has been successfully applied for conditional genome editing in C. elegans, mouse, and rat (Ma et al. 2013; Yang et al. 2013; Liu et al. 2014). More recently, UAS-driven expression of Cas9 in Drosophila has been shown to work with the CRISPR/Cas9 system (Port et al. 2014). Based on the high efficiency of Cas9/gRNA gene targeting, we reasoned that directly disrupting targeted genes at the DNA level would destroy gene function more efficiently than posttranscriptional breakdown of the targeted mRNA mediated by RNAi. Therefore, we combined the CRISPR/Cas9 and Gal4-UAS systems to develop a CRISPR/Cas9-mediated conditional mutagenesis (CMCM) method and systematically investigated whether it can efficiently inactivate gene activity in most Drosophila tissues. This method contains two steps (Figure 1). First, we generated gene-specific gRNAs directed against a number of genes with known phenotypes and made their 10UAS-Cas9/gRNAs transgenic flies. Second, we induced the expression of 10UAS-Cas9/gRNAs driven by the tissue-specific Gal4 lines and investigated the efficiency of gene disruption and associated phenotypes. Using the CMCM system, we tested six genes (y, notch, bam, nos, cid, and ms(3)k81) and achieved highly effective disruption in wing, eye, ovary, and testis tissues. Moreover, side-by-side comparisons of tissue-specific gene disruption showed the power of the CMCM system. In general, it was more efficient and rapid than RNAi approaches and provided a novel genetic tool for studying gene function in specific tissues in Drosophila (Dietzl et al. 2007; Ni et al. 2011).

Bottom Line: Existing transgenic RNA interference (RNAi) methods greatly facilitate functional genome studies via controlled silencing of targeted mRNA in Drosophila.Although the RNAi approach is extremely powerful, concerns still linger about its low efficiency.Here, we developed a CRISPR/Cas9-mediated conditional mutagenesis system by combining tissue-specific expression of Cas9 driven by the Gal4/upstream activating site system with various ubiquitously expressed guide RNA transgenes to effectively inactivate gene expression in a temporally and spatially controlled manner.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Tsinghua University, Beijing 100084, China.

Show MeSH