Improved Mos1-mediated transgenesis in C. elegans

Frøkjær-Jensen, Christian; Davis, Matthew L.; Ailion, Michael; Jørgensen, Erik M.

doi:10.1038/nmeth.1865

Cited by 407 publications

(480 citation statements)

References 6 publications

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Section: Discussionmentioning

confidence: 99%

“…In contrast, a homologous repair event guided by a coinjected DNA plasmid occurs at a lower frequency than repair from an ssOligo template, requiring that both a selection and a counterselection be utilized. That approach leaves a genetic marker near the endogenous locus and is significantly slower than the approach we used, requiring weeks rather than days to recover the insertion (Frokjaer-Jensen et al 2012).Because the insertion strategy permits endogenous changes to occur at some distance from the DSB, it provides flexibility in target selection to avoid complications arising from DNA features needed for nuclease target recognition. Both TALENs and CRISPR/Cas9 have specific, although remarkably minimal, constraints for site recognition.…”

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confidence: 99%

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Precise and Heritable Genome Editing in Evolutionarily Diverse Nematodes Using TALENs and CRISPR/Cas9 to Engineer Insertions and Deletions

et al. 2013

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Exploitation of custom-designed nucleases to induce DNA double-strand breaks (DSBs) at genomic locations of choice has transformed our ability to edit genomes, regardless of their complexity. DSBs can trigger either error-prone repair pathways that induce random mutations at the break sites or precise homology-directed repair pathways that generate specific insertions or deletions guided by exogenously supplied DNA. Prior editing strategies using site-specific nucleases to modify the Caenorhabditis elegans genome achieved only the heritable disruption of endogenous loci through random mutagenesis by error-prone repair. Here we report highly effective strategies using TALE nucleases and RNA-guided CRISPR/Cas9 nucleases to induce error-prone repair and homology-directed repair to create heritable, precise insertion, deletion, or substitution of specific DNA sequences at targeted endogenous loci. Our robust strategies are effective across nematode species diverged by 300 million years, including necromenic nematodes (Pristionchus pacificus), male/female species (Caenorhabditis species 9), and hermaphroditic species (C. elegans). Thus, genome-editing tools now exist to transform nonmodel nematode species into genetically tractable model organisms. We demonstrate the utility of our broadly applicable genome-editing strategies by creating reagents generally useful to the nematode community and reagents specifically designed to explore the mechanism and evolution of X chromosome dosage compensation. By developing an efficient pipeline involving germline injection of nuclease mRNAs and single-stranded DNA templates, we engineered precise, heritable nucleotide changes both close to and far from DSBs to gain or lose genetic function, to tag proteins made from endogenous genes, and to excise entire loci through targeted FLP-FRT recombination. STRATEGIES to engineer heritable, site-directed mutations at endogenous loci have revolutionized our approach toward manipulating and dissecting genome function. Studies of plants and animals alike, whether conducted in whole organisms or cell lines, have benefitted greatly from these genomeediting approaches (Bibikova et al. 2002;Beumer et al. 2006;Doyon et al. 2008;Geurts et al. 2009;Hockemeyer et al. 2009;Holt et al. 2010;Zhang et al. 2010;Hockemeyer et al. 2011;Tesson et al. 2011;Wood et al. 2011;Young et al. 2011;Bedell et al. 2012;Bassett et al. 2013;Cong et al. 2013;Jinek et al. 2013;Mali et al. 2013;Wang et al. 2013;Zu et al. 2013). The most modern tools for modifying complex genomes at single-nucleotide resolution are site-specific nucleases that induce DNA double-strand breaks (DSBs) at specifically designated genomic locations. DSBs trigger repair pathways that can elicit targeted genetic reprogramming, primarily through two mechanisms: error-prone nonhomologous end joining (NHEJ) (Lieber 2010) and precise, homology-directed recombination or repair (HDR) (Chapman et al. 2012). NHEJ rejoins broken ends of chromosomes through an imprecise process that produces nucleo...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Precise and Heritable Genome Editing in Evolutionarily Diverse Nematodes Using TALENs and CRISPR/Cas9 to Engineer Insertions and Deletions

et al. 2013

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“…To maintain a common genetic background, control smn-1(+) animals were similarly derived from +/hT2 parents. Plasmid pHA#582 contains a 1,819-bp fragment corresponding to the smn-1 promoter, coding sequence, and 3′ untranslated region subcloned as an AflII/XhoI product into pCFJ356 (Addgene plasmid 34871) (85). Primers for amplification were as follows: 5-tgatcttaagtctacgagcgacattcatcg and 5-tgatctcgagcagcctctcatcctgattgc.…”

Section: Methodsmentioning

confidence: 99%

“…Primers for amplification were as follows: 5-tgatcttaagtctacgagcgacattcatcg and 5-tgatctcgagcagcctctcatcctgattgc. rtSi9[Cb-unc-119(+)]IV and rtSi10 [smn-1p::smn-1;Cb-unc-119(+)]IV transgenes were generated by Mos1-mediated single-copy insertion (85,86). We injected 50 ng/μL of targeting plasmid (pCFJ356 or pHA#582) into EG6703 [unc-119(ed3)III;cxTi10816 IV] animals along with 50 ng/μL pCFJ601 (eft-3p::Mos1 transposase), 10 ng/μL pGH8 (rab-3p::mCherry), 5 ng/μL pCFJ104 (myo-3p::mCherry), and 2.5 ng/μL pCFJ90 (myo-2p::mCherry).…”

Section: Methodsmentioning

confidence: 99%

Decreased function of survival motor neuron protein impairs endocytic pathways

Dimitriadi

Derdowski

Kalloo

et al. 2016

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

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Spinal muscular atrophy (SMA) is caused by depletion of the ubiquitously expressed survival motor neuron (SMN) protein, with 1 in 40 Caucasians being heterozygous for a disease allele. SMN is critical for the assembly of numerous ribonucleoprotein complexes, yet it is still unclear how reduced SMN levels affect motor neuron function. Here, we examined the impact of SMN depletion in Caenorhabditis elegans and found that decreased function of the SMN ortholog SMN-1 perturbed endocytic pathways at motor neuron synapses and in other tissues. Diminished SMN-1 levels caused defects in C. elegans neuromuscular function, and smn-1 genetic interactions were consistent with an endocytic defect. Changes were observed in synaptic endocytic proteins when SMN-1 levels decreased. At the ultrastructural level, defects were observed in endosomal compartments, including significantly fewer docked synaptic vesicles. Finally, endocytosis-dependent infection by JC polyomavirus (JCPyV) was reduced in human cells with decreased SMN levels. Collectively, these results demonstrate for the first time, to our knowledge, that SMN depletion causes defects in endosomal trafficking that impair synaptic function, even in the absence of motor neuron cell death.endocytic trafficking | survival motor neuron | spinal muscular atrophy | C. elegans | infection S pinal muscular atrophy (SMA) is one of the most severe neuromuscular diseases of childhood, with an incidence of 1 in 10,000 live births and a high carrier frequency of roughly 1 in 40 Caucasians (1-3). SMA is caused by reduced levels of the ubiquitously expressed survival of motor neuron (SMN) protein and results in degeneration of α-spinal cord motor neurons, muscle weakness, and/or death. Two human genes encode the SMN protein, SMN1 and SMN2. SMA alleles arise at relatively high frequency due to small intrachromosomal de novo rearrangements including the SMN1 locus (4). Patients often carry homozygous SMN1 deletions, although missense and nonsense alleles exist (5). Multiple copies of SMN2 rarely compensate for loss of SMN1 due to a C > T nucleotide change in SMN2 exon 7 that perturbs pre-mRNA splicing and results in a truncated protein of diminished function and stability (SMNΔ7) (5-9).SMN has numerous roles and interacts with various proteins, yet it remains unclear which interactions are most pertinent to SMA pathogenesis. As a component of the Gemin complex, SMN is required for biogenesis of small nuclear ribonucleoprotein (snRNP) particles critical for pre-mRNA splicing (10-12). Furthermore, SMN is needed for stress granule formation (13,14), is found in RNP granules moving through neuronal processes, and is part of RNP complexes implicated in synaptic local translation (15)(16)(17)(18)(19)(20). Additional roles for SMN, in transcription (21), in the PTEN-mediated protein synthesis pathway (22), in translational control (23), and in cell proliferation/differentiation (24), have been described. Importantly, no consensus has been reached regarding the cellular and molecular pathways wh...

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“…These reporter constructs are either injected into embryos to generate stable transformants (Venken and Bellen, 2007;Tasic et al, 2011;Frokjaer-Jensen et al, 2012), or electroporated (Funahashi and Nakamura, 2008;Vierra and Irvine, 2012), to observe the transient effect of the construct. In most cases, these small DNA fragments are sufficient to provide coherent functions in vivo.…”

Section: Interactions Between Regulatory Elementsmentioning

confidence: 99%

Multiple layers of complexity in cis‐regulatory regions of developmental genes

Frankel

2012

Developmental Dynamics

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Genomes contain the necessary information to ensure that genes are expressed in the right place, at the right time, and with the proper rate. Metazoan developmental genes often possess long stretches of DNA flanking their coding sequences and/or large introns which contain elements that influence gene expression. Most of these regulatory elements are relatively small and can be studied in isolation. For example, transcriptional enhancers, the elements that generate the expression pattern of a gene, have been traditionally studied with reporter constructs in transgenic animals. These studies have provided and will provide invaluable insights into enhancer evolution and function. However, this experimental approach has its limits; often, enhancer elements do not faithfully recapitulate native expression patterns. This fact suggests that additional information in cis-regulatory regions modulates the activity of enhancers and other regulatory elements. Indeed, recent studies have revealed novel functional aspects at the level of whole cis-regulatory regions. First, the discovery of ''shadow enhancers.'' Second, the ubiquitous interactions between cis-regulatory elements. Third, the notion that some cis-regulatory regions may not function in a modular manner. Last, the effect of chromatin conformation on cis-regulatory activity. In this article, I describe these recent findings and discuss open questions in the field.

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Improved Mos1-mediated transgenesis in C. elegans

Cited by 407 publications

References 6 publications

Precise and Heritable Genome Editing in Evolutionarily Diverse Nematodes Using TALENs and CRISPR/Cas9 to Engineer Insertions and Deletions

Precise and Heritable Genome Editing in Evolutionarily Diverse Nematodes Using TALENs and CRISPR/Cas9 to Engineer Insertions and Deletions

Decreased function of survival motor neuron protein impairs endocytic pathways

Multiple layers of complexity in cis‐regulatory regions of developmental genes

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