Highly Efficient Genome Modifications Mediated by CRISPR/Cas9 in <i>Drosophila</i>

Yu, Zhongsheng; Ren, Mengda; Wang, Zhanxiang; Zhang, Bo; Rong, Yikang S.; Jiao, Renjie; Gao, Guanjun

doi:10.1534/genetics.113.153825

Cited by 336 publications

(315 citation statements)

References 14 publications

(10 reference statements)

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“…In vitro transcription of Cas9 mRNA was performed using the Sp6 mMESSAGE mMACHINE Kit (Ambion), according to Yu et al (2013). In vitro transcription of the designed gRNAs was performed using the RiboMAX Large Scale RNA Production Systems-T7 Kit (Promega).…”

Section: Generation Of Lncrna Knockout Fliesmentioning

confidence: 99%

Critical roles of long noncoding RNAs in Drosophila spermatogenesis

et al. 2016

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Long noncoding RNAs (lncRNAs), a recently discovered class of cellular RNAs, play important roles in the regulation of many cellular developmental processes. Although lncRNAs have been systematically identified in various systems, most of them have not been functionally characterized in vivo in animal models. In this study, we identified 128 testis-specific Drosophila lncRNAs and knocked out 105 of them using an optimized three-component CRISPR/Cas9 system. Among the lncRNA knockouts, 33 (31%) exhibited a partial or complete loss of male fertility, accompanied by visual developmental defects in late spermatogenesis. In addition, six knockouts were fully or partially rescued by transgenes in a trans configuration, indicating that those lncRNAs primarily work in trans. Furthermore, gene expression profiles for five lncRNA mutants revealed that testis-specific lncRNAs regulate global gene expression, orchestrating late male germ cell differentiation. Compared with coding genes, the testis-specific lncRNAs evolved much faster. Moreover, lncRNAs of greater functional importance exhibited higher sequence conservation, suggesting that they are under constant evolutionary selection. Collectively, our results reveal critical functions of rapidly evolving testis-specific lncRNAs in late Drosophila spermatogenesis.

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Section: Generation Of Lncrna Knockout Fliesmentioning

confidence: 99%

Critical roles of long noncoding RNAs in Drosophila spermatogenesis

et al. 2016

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“…In addition, to facilitate prediction and testing of off-target events, we established a searchable database of in silico predicted sgRNAs that includes information about predicted off-target events. Finally, we compared our approach with methodologies recently reported by others (14,16,20). …”

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confidence: 99%

“…The tracrRNA triggers Cas9 nuclease activity and the crRNA guides Cas9 to cleave the specific foreign dsDNA sequence via base-pairing between the crRNA and the target DNA. Importantly, a single-guide RNA (sgRNA, also known as chiRNA), comprising the minimal crRNA and tracrRNA, can function similarly to the crRNA and tracrRNA, thereby providing a simplified method for genome editing (8)(9)(10)(14)(15)(16)(17)(18)(19)(20).…”

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confidence: 99%

Optimized gene editing technology for Drosophila melanogaster using germ line-specific Cas9

Greenleaf

Sun

Housden

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

376

402

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The ability to engineer genomes in a specific, systematic, and costeffective way is critical for functional genomic studies. Recent advances using the CRISPR-associated single-guide RNA system (Cas9/sgRNA) illustrate the potential of this simple system for genome engineering in a number of organisms. Here we report an effective and inexpensive method for genome DNA editing in Drosophila melanogaster whereby plasmid DNAs encoding short sgRNAs under the control of the U6b promoter are injected into transgenic flies in which Cas9 is specifically expressed in the germ line via the nanos promoter. We evaluate the off-targets associated with the method and establish a Web-based resource, along with a searchable, genome-wide database of predicted sgRNAs appropriate for genome engineering in flies. Finally, we discuss the advantages of our method in comparison with other recently published approaches.nanos-Cas9 | HRMA M uch of our knowledge of the mechanisms underlying biological processes relies on genetic approaches, whereby gene activity is perturbed and the phenotypic consequences of perturbation are analyzed in detail. In recent years, several major advances have been made in the design of methods for specifically and efficiently perturbing genomes. Arguably, the most exciting advances rely on the ability to induce double-strand breaks (DSBs) by targeting a nuclease to a specific genomic sequence. Repair of DSBs by the error-prone nonhomologous endjoining (NHEJ) mechanism allows for the recovery of small deletions; moreover, repair of DSBs by homologous recombination (HR) in the presence of a donor template opens the door to a wide range of specifically engineered changes at the targeted site (1).Two nuclease-based systems, the zinc-finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN) systems, work effectively in a number of organisms (2-7). But because these approaches require the production of a construct encoding a unique DNA-binding protein fused to the nuclease domain, they can be both cumbersome and costly. In contrast, the recent approach based on the bacterial CRISPR-associated single-guide RNA (Cas9/sgRNA) system does not require production of specific fusion proteins for each targeted sequence (8-10).Cas9 was first identified in type II Streptococcus pyogenes as an RNA-guided defense system against invading viruses and plasmids (11-13). This adaptive immune-like system contains three components: CRISPR RNA (crRNA), trans-activating CRISPR RNA (tracrRNA), and Cas9. The tracrRNA triggers Cas9 nuclease activity and the crRNA guides Cas9 to cleave the specific foreign dsDNA sequence via base-pairing between the crRNA and the target DNA. Importantly, a single-guide RNA (sgRNA, also known as chiRNA), comprising the minimal crRNA and tracrRNA, can function similarly to the crRNA and tracrRNA, thereby providing a simplified method for genome editing (8)(9)(10)(14)(15)(16)(17)(18)(19)(20).Given the great promise of the Cas9/sgRNA method for genome engineering, we set out to test the sys...

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“…In addition, it is proving an ideal model to study the molecular and physiological role of the intestinal microbiota in post-embryonic development and homeostasis [57][58][59]. Finally, the Drosophila community has generated an extensive experimental toolkit including TF clone libraries [60] and TF overexpression fly lines [61], in vivo transgenic enhancer lines [62,63] as well as genome engineering tools such as CRISPR [64][65][66], rendering the Drosophila gut an ideal model to achieve a level of mechanistic, regulatory understanding that may currently be unattainable for the mammalian gastrointestinal tract.…”

Section: Discussionmentioning

confidence: 99%

Gene regulatory mechanisms underlying the intestinal innate immune response

Meireles-Filho

Deplancke

2017

Current Opinion in Genetics & Development

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In the mammalian gastrointestinal tract, distinct types of cells, including epithelial cells and macrophages, collaborate to eliminate ingested pathogens while striving to preserve the commensal microbiota. The underlying innate immune response is driven by significant gene expression changes in each cell, and recent work has provided novel insights into the gene regulatory mechanisms that mediate such transcriptional changes. These mechanisms differ from those underlying the canonical cellular differentiation model in which a sequential deposition of DNA methylation and histone modification marks progressively restricts the chromatin landscape. Instead, inflammatory macrophages and intestinal epithelial cells appear to largely rely on transcription factors that explore an accessible chromatin landscape to generate dynamic stimulus-specific and spatial-specific physiological responses.

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Highly Efficient Genome Modifications Mediated by CRISPR/Cas9 in Drosophila

Cited by 336 publications

References 14 publications

Critical roles of long noncoding RNAs in Drosophila spermatogenesis

Critical roles of long noncoding RNAs in Drosophila spermatogenesis

Optimized gene editing technology for Drosophila melanogaster using germ line-specific Cas9

Gene regulatory mechanisms underlying the intestinal innate immune response

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