Developmental Modulation of Nonhomologous End Joining in <i>Caenorhabditis elegans</i>

Clejan, Iuval; Boerckel, Julie; Ahmed, Shawn

doi:10.1534/genetics.106.058628

Cited by 107 publications

(132 citation statements)

References 83 publications

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“…In addition, short patches of microhomology .1 nucleotide were apparent at breakpoints for 3/6 clk-2(mn159) deletions and for 4/11 mrt-2 deletions (Tables 4 and 5). Thus, microhomology-mediated end-joining may be responsible for many of the recovered deletions, which would be consistent with observations that lig-4-mediated NHEJ is repressed in the C. elegans germline (Martin et al 2005;Clejan et al 2006).…”

Section: Resultssupporting

confidence: 88%

Mutator Phenotype ofCaenorhabditis elegansDNA Damage Checkpoint Mutants

Harris¹,

Lowden²,

Clejan

et al. 2006

Genetics

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DNA damage response proteins identify sites of DNA damage and signal to downstream effectors that orchestrate either apoptosis or arrest of the cell cycle and DNA repair. The C. elegans DNA damage response mutants mrt-2, hus-1, and clk-2(mn159) displayed 8-to 15-fold increases in the frequency of spontaneous mutation in their germlines. Many of these mutations were small-to medium-sized deletions, some of which had unusual sequences at their breakpoints such as purine-rich tracts or direct or inverted repeats. Although DNA-damage-induced apoptosis is abrogated in the mrt-2, hus-1, and clk-2 mutant backgrounds, lack of the apoptotic branch of the DNA damage response pathway in cep-1/p53, ced-3, and ced-4 mutants did not result in a Mutator phenotype. Thus, DNA damage checkpoint proteins suppress the frequency of mutation by ensuring that spontaneous DNA damage is accurately repaired in C. elegans germ cells. Although DNA damage response defects that predispose humans to cancer are known to result in large-scale chromosome aberrations, our results suggest that small-to medium-sized deletions may also play roles in the development of cancer.

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Section: Resultssupporting

confidence: 88%

Mutator Phenotype ofCaenorhabditis elegansDNA Damage Checkpoint Mutants

Harris¹,

Lowden²,

Clejan

et al. 2006

Genetics

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“…The levels of RAD-51 foci in syp-3(me42); lig-4 followed a trend similar to that observed in either a lig-4 or wild-type background, indicating that NHEJ does not play a primary role in meiotic DSB repair in C. elegans, consistent with previous studies (Martin et al 2005;Clejan et al 2006). However, the contribution of NHEJ to DSB repair becomes more evident when access to sister chromatids as a template for repair is no longer available.…”

Section: Genotypesupporting

confidence: 86%

“…Although NHEJ is not a primary mode of repair in the germline (Martin et al 2005;Clejan et al 2006), NHEJ has recently been observed to participate in meiotic DSB repair in brc-2 mutants where homologous recombination is impaired due to the misregulation of RAD-51 (Martin et al 2005). In this work, we demonstrate that NHEJ can participate in germline DSB repair when both homolog-mediated and sister chromatidmediated recombination are impaired due to synapsis defects and defective sister chromatid cohesion.…”

Section: Discussionmentioning

confidence: 62%

Synapsis-Defective Mutants Reveal a Correlation Between Chromosome Conformation and the Mode of Double-Strand Break Repair DuringCaenorhabditis elegansMeiosis

et al. 2007

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SYP-3 is a new structural component of the synaptonemal complex (SC) required for the regulation of chromosome synapsis. Both chromosome morphogenesis and nuclear organization are altered throughout the germlines of syp-3 mutants. Here, our analysis of syp-3 mutants provides insights into the relationship between chromosome conformation and the repair of meiotic double-strand breaks (DSBs). Although crossover recombination is severely reduced in syp-3 mutants, the production of viable offspring accompanied by the disappearance of RAD-51 foci suggests that DSBs are being repaired in these synapsis-defective mutants. Our studies indicate that once interhomolog recombination is impaired, both intersister recombination and nonhomologous end-joining pathways may contribute to repair during germline meiosis. Moreover, our studies suggest that the conformation of chromosomes may influence the mode of DSB repair employed during meiosis.

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“…Given that both germ cells and the embryo appear competent for homologous repair pathways (Clejan et al 2006), Cas9-induced lesions in the early embryo may be refractory to templated repair simply because the injected (singlestranded) donor DNA is unstable and/or unavailable (as proposed by Kim et al 2014). Additional work will provide further insight into the dynamics of CRISPR/Cas9 genome editing in the germline of C. elegans.…”

Section: Homozygous Oligonucleotide-templated Conversion Is Rarementioning

confidence: 99%

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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Developmental Modulation of Nonhomologous End Joining in Caenorhabditis elegans

Cited by 107 publications

References 83 publications

Mutator Phenotype ofCaenorhabditis elegansDNA Damage Checkpoint Mutants

Mutator Phenotype ofCaenorhabditis elegansDNA Damage Checkpoint Mutants

Synapsis-Defective Mutants Reveal a Correlation Between Chromosome Conformation and the Mode of Double-Strand Break Repair DuringCaenorhabditis elegansMeiosis

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

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