Efficient gene targeting in
            <i>Drosophila</i>
            by direct embryo injection with zinc-finger nucleases

Beumer, Kelly J.; Trautman, Jonathan K.; Bozas, Ana; Liu, Ji‐Long; Rutter, Jared; Gall, Joseph G.; Carroll, Dana

doi:10.1073/pnas.0810475105

Cited by 279 publications

(270 citation statements)

References 52 publications

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“…These findings indicate that spontaneous homologous recombination is a rare event in the sea urchin embryo and that a ZFN-induced DSB at the target site stimulates HDR, resulting in the induction of targeted transgene insertion at a detectable level. This result is in agreement with earlier work using Drosophila and mice (7,14), suggesting that ZFN-mediated targeted insertion is feasible in model animals, although the efficiency of the insertion may depend on the species. Addition of ZFN target sites at both ends of the targeting donor cassette significantly increased the efficiency of ZFN-mediated targeted transgene insertion.…”

Section: Discussionsupporting

confidence: 82%

“…It has been reported that DNA ligase IV, a major component of the NHEJ pathway, forms a complex with XRCC4 and seals DNA ends (22). Moreover, DSB repair can be biased toward HDR by disrupting the function of DNA ligase IV, resulting in an increase in the efficiency of targeted gene modification (7). Therefore, we hypothesized that in the sea urchin, inhibition of DNA ligase IV through the introduction of DN-lig4 mRNA increases the propensity to repair ZFN-induced DSBs through HDR, resulting in enhanced efficiency of ZFN-mediated targeted transgene insertion.…”

Section: Discussionmentioning

confidence: 99%

“…Therefore, ZFNs can introduce a site-specific insertion or deletion at the DSB site after NHEJ repair (9,11). Alternatively, ZFNs can produce defined genetic modifications, including the insertion of a reporter gene, near the site of the DSB by HDR using an exogenous targeting donor construct (7,11). In animals, it has been noted that ZFN-induced DSBs are mainly repaired by NHEJ, and therefore ZFN-mediated targeted gene correction and transgene insertion are considered to be challenging (7,14).…”

mentioning

confidence: 99%

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Zinc-finger nuclease-mediated targeted insertion of reporter genes for quantitative imaging of gene expression in sea urchin embryos

Ochiai

Sakamoto

Fujita

et al. 2012

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

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To understand complex biological systems, such as the development of multicellular organisms, it is important to characterize the gene expression dynamics. However, there is currently no universal technique for targeted insertion of reporter genes and quantitative imaging in multicellular model systems. Recently, genome editing using zinc-finger nucleases (ZFNs) has been reported in several models. ZFNs consist of a zinc-finger DNA-binding array with the nuclease domain of the restriction enzyme FokI and facilitate targeted transgene insertion. In this study, we successfully inserted a GFP reporter cassette into the HpEts1 gene locus of the sea urchin, Hemicentrotus pulcherrimus. We achieved this insertion by injecting eggs with a pair of ZFNs for HpEts1 with a targeting donor construct that contained ∼1-kb homology arms and a 2A-histone H2B-GFP cassette. We increased the efficiency of the ZFNmediated targeted transgene insertion by in situ linearization of the targeting donor construct and cointroduction of an mRNA for a dominant-negative form of HpLig4, which encodes the H. pulcherrimus homolog of DNA ligase IV required for error-prone nonhomologous end joining. We measured the fluorescence intensity of GFP at the single-cell level in living embryos during development and found that there was variation in HpEts1 expression among the primary mesenchyme cells. These findings demonstrate the feasibility of ZFN-mediated targeted transgene insertion to enable quantification of the expression levels of endogenous genes during development in living sea urchin embryos.live imaging | quantitative biology

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Section: Discussionsupporting

confidence: 82%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Zinc-finger nuclease-mediated targeted insertion of reporter genes for quantitative imaging of gene expression in sea urchin embryos

Ochiai

Sakamoto

Fujita

et al. 2012

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

View full text Add to dashboard Cite

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“…This latter approach ensures that DNA is cleaved only when ZFNs for the left and right halfsites are positioned at the appropriate target sequence on the chromosome. Limited expression of ZFNs either by transient DNA delivery, injected RNA, or inducible expression systems has also been suggested as a strategy to reduce undesired toxic effects (32,33,36).…”

Section: Discussionmentioning

confidence: 99%

High frequency targeted mutagenesis in Arabidopsis thaliana using zinc finger nucleases

Zhang

Maeder

Unger-Wallace

et al. 2010

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

343

228

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We report here an efficient method for targeted mutagenesis of Arabidopsis genes through regulated expression of zinc finger nucleases (ZFNs)-enzymes engineered to create DNA doublestrand breaks at specific target loci. ZFNs recognizing the Arabidopsis ADH1 and TT4 genes were made by Oligomerized Pool ENgineering (OPEN)-a publicly available, selection-based platform that yields high quality zinc finger arrays. The ADH1 and TT4 ZFNs were placed under control of an estrogen-inducible promoter and introduced into Arabidopsis plants by floral-dip transformation. Primary transgenic Arabidopsis seedlings induced to express the ADH1 or TT4 ZFNs exhibited somatic mutation frequencies of 7% or 16%, respectively. The induced mutations were typically insertions or deletions (1-142 bp) that were localized at the ZFN cleavage site and likely derived from imprecise repair of chromosome breaks by nonhomologous end-joining. Mutations were transmitted to the next generation for 69% of primary transgenics expressing the ADH1 ZFNs and 33% of transgenics expressing the TT4 ZFNs. Furthermore, ≈20% of the mutant-producing plants were homozygous for mutations at ADH1 or TT4, indicating that both alleles were disrupted. ADH1 and TT4 were chosen as targets for this study because of their selectable or screenable phenotypes (adh1, allyl alcohol resistance; tt4, lack of anthocyanins in the seed coat). However, the high frequency of observed ZFNinduced mutagenesis suggests that targeted mutations can readily be recovered by simply screening progeny of primary transgenic plants by PCR and DNA sequencing. Taken together, our results suggest that it should now be possible to obtain mutations in any Arabidopsis target gene regardless of its mutant phenotype.nonhomologous end-joining | gene knockout | alcohol dehydrogenase | chalcone synthase

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“…A y w stock was used as control. Two coilin null lines, coil 199 and coil 203 , were generated by site-specific mutation using zincfinger nucleases (Beumer et al, 2008;Liu et al, 2009). Drosophila S2 cells were used as an additional wild-type control.…”

Section: Fliesmentioning

confidence: 99%

Small Cajal Body–specific RNAs ofDrosophilaFunction in the Absence of Cajal Bodies

Deryusheva

Gall

2009

MBoC

Self Cite

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During their biogenesis small nuclear RNAs (snRNAs) undergo multiple covalent modifications that require guide RNAs to direct methylase and pseudouridylase enzymes to the appropriate nucleotides. Because of their localization in the nuclear Cajal body (CB), these guide RNAs are known as small CB-specific RNAs (scaRNAs). Using a fluorescent primer extension technique, we mapped the modified nucleotides in Drosophila U1, U2, U4, and U5 snRNAs. By fluorescent in situ hybridization (FISH) we showed that seven Drosophila scaRNAs are concentrated in easily detectable CBs. We used two assays based on Xenopus oocyte nuclei to demonstrate that three of these Drosophila scaRNAs do, in fact, function as guide RNAs. In flies null for the CB marker protein coilin, CBs are absent and there are no localized FISH signals for the scaRNAs. Nevertheless, biochemical experiments show that scaRNAs are present at normal levels and snRNAs are properly modified. Our experiments demonstrate that several scaRNAs are concentrated as expected in the CBs of wild-type Drosophila, but they function equally well in the nucleoplasm of mutant flies that lack CBs. We propose that the snRNA modification machinery is not limited to CBs, but is dispersed throughout the nucleoplasm of cells in general. INTRODUCTIONThe two major ribosomal RNAs (rRNAs) and the spliceosomal small nuclear RNAs (snRNAs) U1, U2, U4, U5, and U6 contain many posttranscriptionally modified nucleotides, which are crucial for RNA-RNA and RNA-protein interactions, as well as spliceosome function (Yu et al., 1998; Yu, 2004, 2007). The most abundant modifications in both rRNAs and snRNAs are 2Ј-O-methylation and pseudouridylation, which are directed by small box C/D and box H/ACA guide RNAs, respectively. Each guide RNA molecule is associated with a set of four core proteins: box C/D RNAs form RNP particles with fibrillarin (the methyltransferase), Nop56, Nop58, and a 15.5-kDa protein, whereas box H/ACA RNAs associate with NAP57/dyskerin (the pseudouridine synthase), GAR1, NHP2, and NOP10 proteins (reviewed in Yu et al., 2004;Matera et al., 2007). Most guide RNAs are concentrated in the nucleolus, where they are involved in posttranscriptional modifications of rRNA. Because of their localization, they are referred to as small nucleolar RNAs (snoRNAs). However, the guide RNAs that mediate modification of the snRNAs are preferentially found in another nuclear organelle, the Cajal body (CB). These guide RNAs are called small CB-specific RNAs (scaRNAs).The first scaRNA to be studied in detail was U85 scaRNA (Jády and Kiss, 2001;Darzacq et al., 2002). U85 is a remarkable double guide RNA that contains both box C/D and box H/ACA motifs. In a set of detailed experiments it was shown that human U85 scaRNA guides both 2Ј-O-methylation at C45 and pseudouridylation at U46 in U5 snRNA. Modification takes place in the nucleus after import of the U5 snRNA from the cytoplasm . CB localization of U85 scaRNA was originally demonstrated by fluorescent in situ hybridization (FISH) in mammalian and ...

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Efficient gene targeting in Drosophila by direct embryo injection with zinc-finger nucleases

Cited by 279 publications

References 52 publications

Zinc-finger nuclease-mediated targeted insertion of reporter genes for quantitative imaging of gene expression in sea urchin embryos

Zinc-finger nuclease-mediated targeted insertion of reporter genes for quantitative imaging of gene expression in sea urchin embryos

High frequency targeted mutagenesis in Arabidopsis thaliana using zinc finger nucleases

Small Cajal Body–specific RNAs ofDrosophilaFunction in the Absence of Cajal Bodies

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