Dataset Information

A multiplexed DNA FISH strategy for assessing genome architecture in Caenorhabditis elegans.

ABSTRACT: Eukaryotic DNA is highly organized within nuclei and this organization is important for genome function. Fluorescent in situ hybridization (FISH) approaches allow 3D architectures of genomes to be visualized. Scalable FISH technologies, which can be applied to whole animals, are needed to help unravel how genomic architecture regulates, or is regulated by, gene expression during development, growth, reproduction, and aging. Here, we describe a multiplexed DNA FISH Oligopaint library that targets the entire Caenorhabditis elegans genome at chromosome, three megabase, and 500 kb scales. We describe a hybridization strategy that provides flexibility to DNA FISH experiments by coupling a single primary probe synthesis reaction to dye conjugated detection oligos via bridge oligos, eliminating the time and cost typically associated with labeling probe sets for individual experiments. The approach allows visualization of genome organization at varying scales in all/most cells across all stages of development in an intact animal model system.

SUBMITTER: Fields BD

PROVIDER: S-EPMC6516958 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Publications

A multiplexed DNA FISH strategy for assessing genome architecture in <i>Caenorhabditis elegans</i>.

Fields Brandon D BD Nguyen Son C SC Nir Guy G Kennedy Scott S

eLife 20190514

Eukaryotic DNA is highly organized within nuclei and this organization is important for genome function. Fluorescent <i>in situ</i> hybridization (FISH) approaches allow 3D architectures of genomes to be visualized. Scalable FISH technologies, which can be applied to whole animals, are needed to help unravel how genomic architecture regulates, or is regulated by, gene expression during development, growth, reproduction, and aging. Here, we describe a multiplexed DNA FISH Oligopaint library that ...[more]

PMID: 31084706

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Project description:Null mutants are essential for analyzing gene function. Here, we describe a simple and efficient method to generate Caenorhabditis elegans null mutants using CRISPR/Cas9 and short single stranded DNA oligo repair templates to insert a universal 43-nucleotide-long knock-in cassette (STOP-IN) into the early exons of target genes. This STOP-IN cassette has stop codons in all three reading frames and leads to frameshifts, which will generate putative null mutations regardless of the reading frame of the insertion position in exons. The STOP-IN cassette also contains an exogenous Cas9 target site that allows further genome editing and provides a unique sequence that simplifies the identification of successful insertion events via PCR. As a proof of concept, we inserted the STOP-IN cassette at a Cas9 target site in aex-2 to generate new putative null alleles by injecting preassembled Cas9 ribonucleoprotein and a short synthetic single stranded DNA repair template containing the STOP-IN cassette and two ?35-nucleotide-long homology arms identical to the sequences flanking the Cas9 cut site. We showed that these new aex-2 alleles phenocopied an existing loss-of-function allele of aex-2 We further showed that the new aex-2 null alleles could be reverted back to the wild-type sequence by targeting the exogenous Cas9 cut site included in the STOP-IN cassette and providing a single stranded wild-type DNA repair oligo. We applied our STOP-IN method to generate new putative null mutants for 20 additional genes, including three pharyngeal muscle-specific genes (clik-1, clik-2, and clik-3), and reported a high insertion rate (46%) based on the animals we screened. We showed that null mutations of clik-2 cause recessive lethality with a severe pumping defect and clik-3 null mutants have a mild pumping defect, while clik-1 is dispensable for pumping. We expect that the knock-in method using the STOP-IN cassette will facilitate the generation of new null mutants to understand gene function in C. elegans and other genetic model organisms.

Dataset Information

A multiplexed DNA FISH strategy for assessing genome architecture in Caenorhabditis elegans.

Publications

A multiplexed DNA FISH strategy for assessing genome architecture in <i>Caenorhabditis elegans</i>.

Similar Datasets

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