Protection from Feed-Forward Amplification in an Amplified RNAi Mechanism

Pak, Julia; Maniar, Jay M.; Mello, Cecilia C.; Fire, Andrew

doi:10.1016/j.cell.2012.10.022

Cited by 80 publications

(84 citation statements)

References 57 publications

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“…The rde-1(AAA) plasmid used for integration in Figure ( Pak et al 2012). The rde-1(AAA) PCR product was generated with primers AF-JA-21/82 using pJA32, a derivative of pJP44.3.1, as a substrate.…”

Section: Strainsmentioning

confidence: 99%

“…Donor oligonucleotides encompassed 50 nt of flanking homology on either side. Oligonucleotide preparations (IDT) were dissolved to a final concentration of 10 mM and used in injection mixes at 20 ng/ml without further purification.The rde-1(AAA) plasmid used for integration in Figure ( Pak et al 2012). The rde-1(AAA) PCR product was generated with primers AF-JA-21/82 using pJA32, a derivative of pJP44.3.1, as a substrate.…”

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Efficient Marker-Free Recovery of Custom Genetic Modifications with CRISPR/Cas9 in Caenorhabditis elegans

et al. 2014

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Facilitated by recent advances using CRISPR/Cas9, genome editing technologies now permit custom genetic modifications in a wide variety of organisms. Ideally, modified animals could be both efficiently made and easily identified with minimal initial screening and without introducing exogenous sequence at the locus of interest or marker mutations elsewhere. To this end, we describe a coconversion strategy, using CRISPR/Cas9 in which screening for a dominant phenotypic oligonucleotide-templated conversion event at one locus can be used to enrich for custom modifications at another unlinked locus. After the desired mutation is identified among the F 1 progeny heterozygous for the dominant marker mutation, F 2 animals that have lost the marker mutation are picked to obtain the desired mutation in an unmarked genetic background. We have developed such a coconversion strategy for Caenorhabditis elegans, using a number of dominant phenotypic markers. Examining the coconversion at a second (unselected) locus of interest in the marked F 1 animals, we observed that 14-84% of screened animals showed homologous recombination. By reconstituting the unmarked background through segregation of the dominant marker mutation at each step, we show that custom modification events can be carried out recursively, enabling multiple mutant animals to be made. While our initial choice of a coconversion marker [rol-6(su1006)] was readily applicable in a single round of coconversion, the genetic properties of this locus were not optimal in that CRISPR-mediated deletion mutations at the unselected rol-6 locus can render a fraction of coconverted strains recalcitrant to further rounds of similar mutagenesis. An optimal marker in this sense would provide phenotypic distinctions between the desired mutant/+ class and alternative +/+, mutant/null, null/null, and null/+ genotypes. Reviewing dominant alleles from classical C. elegans genetics, we identified one mutation in dpy-10 and one mutation in sqt-1 that meet these criteria and demonstrate that these too can be used as effective conversion markers. Coconversion was observed using a variety of donor molecules at the second (unselected) locus, including oligonucleotides, PCR products, and plasmids. We note that the coconversion approach described here could be applied in any of the variety of systems where suitable coconversion markers can be identified from previous intensive genetic analyses of gain-of-function alleles.T YPE II CRISPR/Cas9 bacterial immunity systems provide programmable DNA endonuclease activities that have recently revolutionized genome editing in a wide range of organisms (Wang et al. 1999; Chiu et al. 2013;Cho et al. 2013;Dicarlo et al. 2013;Friedland et al. 2013;Gratz et al. 2013;Jiang et al. 2013;Katic and Großhans 2013;Li et al. 2013;Lo et al. 2013;Nekrasov et al. 2013;Kim et al. 2014;Zhao et al. 2014). Recognition by the Cas9 protein entails two sequence elements in the target: a protospacer adjacent motif (PAM) (NGG for Streptococcus pyogenes Cas9) and a re...

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Section: Strainsmentioning

confidence: 99%

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Efficient Marker-Free Recovery of Custom Genetic Modifications with CRISPR/Cas9 in Caenorhabditis elegans

et al. 2014

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“…RRF-1 is a somatic RNA-dependent RNA polymerase acting upstream but necessary to feed the nuclear RNAi pathway. RRF-1 is guided by the RDE-1/siRNA complex to complementary mRNA targets to generate secondary siRNAs (Pak et al 2012), which bind to NRDE-3 to be escorted to the nucleus (Guang et al 2008).…”

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confidence: 99%

Nuclear RNAi Contributes to the Silencing of Off-Target Genes and Repetitive Sequences in Caenorhabditis elegans

Zhou¹,

Xu²,

Hui

et al. 2014

Genetics

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Small RNAs recognize, bind, and regulate other complementary cellular RNAs. The introduction of small RNAs to eukaryotic cells frequently results in unintended silencing of related, but not identical, RNAs: a process termed off-target gene silencing. Off-target gene silencing is one of the major concerns during the application of small RNA-based technologies for gene discovery and the treatment of human disease. Off-target gene silencing is commonly thought to be due to inherent biochemical limitations of the RNAi machinery. Here we show that following the introduction of exogenous sources of double-stranded RNA, the nuclear RNAi pathway, but not its cytoplasmic counterparts, is the primary source of off-target silencing in Caenorhabditis elegans. In addition, we show that during the normal course of growth and development the nuclear RNAi pathway regulates repetitive gene families. Therefore, we speculate that RNAi off-target effects might not be "mistakes" but rather an intentional and genetically programmed aspect of small RNA-mediated gene silencing, which might allow small RNAs to silence rapidly evolving parasitic nucleic acids. Finally, reducing off-target effects by manipulating the nuclear RNAi pathway in vivo might improve the efficacy of small RNAbased technologies.

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“…Primary siRNAs are low in abundance, have Dicerdependent biogenesis, and are 26 nucleotides long with a 59 guanine (26G-siRNAs) Grishok et al 2001;Ketting et al 2001;Knight and Bass 2001;Han et al 2009;Pavelec et al 2009;Vasale et al 2010). Through an unknown mechanism, 26G-siRNAs stimulate the production of highly abundant siRNAs that are 22 nucleotides long with a 59 guanine (22G-siRNAs), and are synthesized through the action of RNA-dependent RNA polymerases (RdRPs) (Smardon et al 2000;Ketting et al 2001;Knight and Bass 2001;Simmer et al 2002;Ambros et al 2003;Maine et al 2005;Vought et al 2005;Aoki et al 2007;Pak and Fire 2007;She et al 2009;Gent et al 2010;Vasale et al 2010;Pak et al 2012). In addition, a group of proteins called the Mutators were shown to play a role in siRNA amplification of both 26G-and 22G-siRNAs classes Phillips et al 2012).…”

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Endogenous RNAi Pathways Are Required in Neurons for Dauer Formation in Caenorhabditis elegans

Bharadwaj¹,

Hall

2017

Genetics

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Animals can adapt to unfavorable environments through changes in physiology or behavior. In the nematode, Caenorhabditis elegans, environmental conditions perceived early in development determine whether the animal enters either the reproductive cycle, or enters into an alternative diapause stage named dauer. Here, we show that endogenous RNAi pathways play a role in dauer formation in crowding (high pheromone), starvation, and high temperature conditions. Disruption of the Mutator proteins or the nuclear Argonaute CSR-1 result in differential dauer-deficient phenotypes that are dependent upon the experienced environmental stress. We provide evidence that the RNAi pathways function in chemosensory neurons for dauer formation, upstream of the TGF-b and insulin signaling pathways. In addition, we show that Mutator MUT-16 expression in a subset of individual pheromone-sensing neurons is sufficient for dauer formation in high pheromone conditions, but not in starvation or high temperature conditions. Furthermore, we also show that MUT-16 and CSR-1 are required for expression of a subset of G proteins with functions in the detection of pheromone components. Together, our data suggest a model where Mutator-amplified siRNAs that associate with the CSR-1 pathway promote expression of genes required for the detection and signaling of environmental conditions to regulate development and behavior in C. elegans. This study highlights a mechanism whereby RNAi pathways mediate the link between environmental stress and adaptive phenotypic plasticity in animals.

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Protection from Feed-Forward Amplification in an Amplified RNAi Mechanism

Cited by 80 publications

References 57 publications

Efficient Marker-Free Recovery of Custom Genetic Modifications with CRISPR/Cas9 in Caenorhabditis elegans

Efficient Marker-Free Recovery of Custom Genetic Modifications with CRISPR/Cas9 in Caenorhabditis elegans

Nuclear RNAi Contributes to the Silencing of Off-Target Genes and Repetitive Sequences in Caenorhabditis elegans

Endogenous RNAi Pathways Are Required in Neurons for Dauer Formation in Caenorhabditis elegans

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