Heritable genome editing in C. elegans via a CRISPR-Cas9 system

Friedland, Ari E.; Tzur, Yonatan B; Esvelt, Kevin M.; Colaiácovo, Mónica P.; Church, George M.; Calarco, John A.

doi:10.1038/nmeth.2532

Cited by 838 publications

(732 citation statements)

References 25 publications

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“…A common approach for this relies on RNAi, which may, however, not always work with high efficiency, especially if genes are expressed in specific cell types such as neurons [78,79]. Another approach is to use knock-out mutants, which are already available for a large number of C. elegans genes [80] and which can be produced through application of CRISPR/Cas technology [81,82]. On the other hand, additional information on antimicrobial functions can be obtained through analysis of synthesized proteins and peptides.…”

Section: Future Challenges: Functional Evidence For Worm Immune Effecmentioning

confidence: 99%

Antimicrobial effectors in the nematode Caenorhabditis elegans : an outgroup to the Arthropoda

Dierking

Yang

Schulenburg

2016

Phil. Trans. R. Soc. B

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One contribution of 13 to a theme issue 'Evolutionary ecology of arthropod antimicrobial peptides'. Nematodes and arthropods likely form the taxon Ecdysozoa. Information on antimicrobial effectors from the model nematode Caenorhabditis elegans may thus shed light on the evolutionary origin of these defences in arthropods. This nematode species possesses an extensive armory of putative antimicrobial effector proteins, such as lysozymes, caenopores (or saposin-like proteins), defensin-like peptides, caenacins and neuropeptide-like proteins, in addition to the production of reactive oxygen species and autophagy. As C. elegans is a bacterivore that lives in microbe-rich environments, some of its effector peptides and proteins likely function in both digestion of bacterial food and pathogen elimination. In this review, we provide an overview of C. elegans immune effector proteins and mechanisms. We summarize the experimental evidence of their antimicrobial function and involvement in the response to pathogen infection. We further evaluate the microbe-induced expression of effector genes using WormExp, a recently established database for C. elegans gene expression analysis. We emphasize the need for further analysis at the protein level to demonstrate an antimicrobial activity of these molecules both in vitro and in vivo.This article is part of the themed issue 'Evolutionary ecology of arthropod antimicrobial peptides'.

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Section: Future Challenges: Functional Evidence For Worm Immune Effecmentioning

confidence: 99%

Antimicrobial effectors in the nematode Caenorhabditis elegans : an outgroup to the Arthropoda

Dierking

Yang

Schulenburg

2016

Phil. Trans. R. Soc. B

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“…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).…”

mentioning

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.

368

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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“…Compared with the zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN), which have been used for genome editing [1], the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/ CRISPR-associated (Cas) system has emerged as a new powerful tool for genome modifications. It has recently been adopted for genome editing in human cell lines [2][3][4], mouse [5], zebrafish [6], C. elegans [7][8][9][10][11][12], and plants [13].…”

mentioning

confidence: 99%

“…We used the Peft-3::cas9::SV40 NLS::tbb-2 3′ UTR vector and sgRNA driven by the C. elegans U6 promoter to ensure stable and efficient expression of the cas9 gene and sgRNA in the C. elegans germline [7]. sgRNAs were designed to target sequences of interest in the form of G/A(N) 19 , which precede the NGG protospacer-adjacent motif (PAM) in the target sites [2][3][4].…”

mentioning

confidence: 99%

Oligonucleotide-based targeted gene editing in C. elegans via the CRISPR/Cas9 system

Zhao

Zhang

et al. 2014

Cell Res

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Heritable genome editing in C. elegans via a CRISPR-Cas9 system

Cited by 838 publications

References 25 publications

Antimicrobial effectors in the nematode Caenorhabditis elegans : an outgroup to the Arthropoda

Antimicrobial effectors in the nematode Caenorhabditis elegans : an outgroup to the Arthropoda

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

Oligonucleotide-based targeted gene editing in C. elegans via the CRISPR/Cas9 system

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