Efficient Gene Knock-out and Knock-in with Transgenic Cas9 in <i>Drosophila</i>

Xue, Zhaoyu; Ren, Mengda; Wu, Menghua; Dai, Junbiao; Rong, Yikang S.; Gao, Guanjun

doi:10.1534/g3.114.010496

Cited by 67 publications

(75 citation statements)

References 18 publications

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“…We and others have recently developed the CRISPR/Cas9 system in Drosophila (Bassett et al, 2013; Gratz et al, 2013; Kondo and Ueda, 2013; Ren et al, 2013; Yu et al, 2013; Gratz et al, 2014; Port et al, 2014; Xue et al, 2014; Yu et al, 2014). However, there have been no systematic studies evaluating the efficiency and specificity in this system.…”

Section: Introductionmentioning

confidence: 99%

Enhanced Specificity and Efficiency of the CRISPR/Cas9 System with Optimized sgRNA Parameters in Drosophila

et al. 2014

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Summary The CRISPR/Cas9 system has recently emerged as a powerful tool for functional genomic studies in Drosophila melanogaster. However, sgRNA parameters affecting the specificity and efficiency of the system in flies are still not clear. Here, we found that off-target effects did not occur in regions of genomic DNA with three or more nucleotide mismatches to sgRNAs. Importantly, we document for the first time a strong positive correlation between mutagenesis efficiency and sgRNA GC content of the six protospacer adjacent motif-proximal nucleotides (PAMPNs). Furthermore, by injecting well-designed sgRNA plasmids at the optimal concentration we determined, we could efficiently generate mutations in four genes in one step. Finally, we generated null alleles of HP1a using optimized parameters through homology-directed repair, and achieved an overall mutagenesis rate significantly higher than previously reported. Our work presents the most comprehensive optimization of sgRNA and promises to vastly simplify CRISPR/Cas9 experiments in Drosophila.

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Section: Introductionmentioning

confidence: 99%

Enhanced Specificity and Efficiency of the CRISPR/Cas9 System with Optimized sgRNA Parameters in Drosophila

et al. 2014

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“…The recent introduction of CRISPR/Cas has made GT via direct injection of donor DNAs plus supporting reagents (e.g., guide RNA) into early embryos possible in Drosophila (BaenaLopez et al 2013; Gratz et al 2013;Bassett and Liu 2014;Port et al 2014;Xue et al 2014;Yu et al 2014). In addition to rapid turnover, direct embryo injection allows easy adoption of diverse GT strategies.…”

Section: Discussionmentioning

confidence: 99%

“…With CRISPR/Cas9, direct embryo injection for ends-out GT in Drosophila has been demonstrated (Baena-Lopez et al 2013;Gratz et al 2013;Port et al 2014;Xue et al 2014;Yu et al 2014) (Figure 1B). This saves the 2 months needed for the initial transgenesis of the donor DNA and may eliminate the need for complex selections.…”

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confidence: 99%

An Enhanced Gene Targeting Toolkit forDrosophila: Golic+

et al. 2015

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Ends-out gene targeting allows seamless replacement of endogenous genes with engineered DNA fragments by homologous recombination, thus creating designer "genes" in the endogenous locus. Conventional gene targeting in Drosophila involves targeting with the preintegrated donor DNA in the larval primordial germ cells. Here we report gene targeting during oogenesis with lethality inhibitor and CRISPR/Cas (Golic+), which improves on all major steps in such transgene-based gene targeting systems. First, donor DNA is integrated into precharacterized attP sites for efficient flip-out. Second, FLP, I-SceI, and Cas9 are specifically expressed in cystoblasts, which arise continuously from female germline stem cells, thereby providing a continual source of independent targeting events in each offspring. Third, a repressorbased lethality selection is implemented to facilitate screening for correct targeting events. Altogether, Golic+ realizes high-efficiency ends-out gene targeting in ovarian cystoblasts, which can be readily scaled up to achieve high-throughput genome editing.

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“…Thus, the targeted modification must occur in the germ cells of the injected organism and be compatible with viable progeny. In fruit flies, this process is facilitated by using transgenic animals that selectively express Cas9 in the germline (Kondo and Ueda 2013;Ren et al 2013;Gratz et al 2014;Sebo et al 2014;Xue et al 2014). The frequency of targeted events is increased using Cas9 transgenic animals, most likely because more consistent levels of Cas9 are achieved with a stably integrated transgene than with injected plasmid, mRNA, or protein.…”

Section: Practical Considerations Of Using Rgns In Developing Organismsmentioning

confidence: 99%

“…Transgenic expression of the gRNA has also been demonstrated to increase the frequency of targeted events in fruit flies (Kondo and Ueda 2013;Port et al 2014;Xue et al 2014). The expression of Cas9 can be restricted to the germline by placing it under the control of tissuespecific regulatory sequences.…”

Section: Practical Considerations Of Using Rgns In Developing Organismsmentioning

confidence: 99%

A CRISPR view of development

et al. 2014

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The CRISPR (clustered regularly interspaced short palindromic repeat)–Cas9 (CRISPR-associated nuclease 9) system is poised to transform developmental biology by providing a simple, efficient method to precisely manipulate the genome of virtually any developing organism. This RNA-guided nuclease (RGN)-based approach already has been effectively used to induce targeted mutations in multiple genes simultaneously, create conditional alleles, and generate endogenously tagged proteins. Illustrating the adaptability of RGNs, the genomes of >20 different plant and animal species as well as multiple cell lines and primary cells have been successfully modified. Here we review the current and potential uses of RGNs to investigate genome function during development.

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Efficient Gene Knock-out and Knock-in with Transgenic Cas9 in Drosophila

Cited by 67 publications

References 18 publications

Enhanced Specificity and Efficiency of the CRISPR/Cas9 System with Optimized sgRNA Parameters in Drosophila

Enhanced Specificity and Efficiency of the CRISPR/Cas9 System with Optimized sgRNA Parameters in Drosophila

An Enhanced Gene Targeting Toolkit forDrosophila: Golic+

A CRISPR view of development

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