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mrt-2 encodes MRT-2, the RAD1 subunit of the PCNA-like 9-1-1 sliding clamp that is loaded onto damaged DNA by the RAD17 clamp loader HPR-17. In the germ line it is required for both branches of the DNA damage response: apoptosis after irradiation, and cell cycle arrest in mitotic germ cells after irradiation or hydroxyurea.
The defect is progressive rather than acute: telomeres shorten at about 130 base pairs per generation, in a single pathway with telomerase, so lines are fertile for a while and then become sterile, typically between generation F10 and F28, with end-to-end fusions and a rising incidence of males. The same strains show a mutator phenotype, whereas apoptotic-branch mutants do not. CD BioSciences delivers custom mrt-2 alleles with full genotype verification.
| Gene symbol | mrt-2 (the RAD1 subunit of the PCNA-like 9-1-1 sliding clamp of C. elegans, encoded by Y41C4A.14 on chromosome III, loaded onto single-stranded DNA at damage sites by the RAD17 clamp loader HPR-17 together with its replication factor C partners) |
|---|---|
| Protein product | MRT-2 — RAD1 subunit of the 9-1-1 checkpoint sliding clamp, orthologous to human RAD1 |
| Mammalian homolog | RAD1 |
| Biological role | MRT-2 forms the heterotrimeric 9-1-1 clamp with HUS-1 and RAD-9, a ring-shaped complex structurally related to PCNA that is loaded at sites of damage independently of the ATM and ATR sensor kinases and then amplifies the signal through them. In the worm germ line it is required for both outputs of the response: mrt-2 mutants fail to mount the apoptotic response to gamma irradiation and fail to arrest the cell cycle in mitotic germ cells after irradiation or hydroxyurea. The same complex is needed at chromosome ends, where it acts in a single pathway with the telomerase catalytic subunit TRT-1 rather than in a parallel one. Because the clamp suppresses the consequences of endogenous damage, losing it produces a mutator phenotype: mutant germ lines accumulate small- to medium-sized deletions at elevated frequency, often with purine-rich tracts or direct and inverted repeats at the breakpoints. |
| Loss-of-function phenotype | Loss of MRT-2 abolishes DNA-damage-induced apoptosis and the mitotic germ-cell arrest response, and produces a progressive, trans-generational phenotype rather than an acute one. Telomeres shorten at roughly 130 base pairs per generation — statistically indistinguishable from the rate in trt-1 mutants, and not additive in the double mutant — so lines remain fertile for a period and then become sterile, in the reference allele between about generation F10 and F28, with end-to-end chromosome fusions and a high-incidence-of-males phenotype that worsens in later generations as X-autosome fusions accumulate. The mutator phenotype appears in the same strains, whereas mutants that lack only the apoptotic branch, such as cep-1, ced-3 and ced-4, do not show it, which separates genome maintenance from cell death cleanly. |
| Why edit it | MRT-2 is the right choice when the question concerns the sensor end of the response rather than the transcriptional output, and its phenotype is unusually informative because it is time-dependent: the animal is superficially healthy and fertile for many generations before the line collapses. That makes it the standard model for studying how telomerase and the damage clamp cooperate at chromosome ends, and it also provides the cleanest demonstration that checkpoint proteins suppress spontaneous mutation by ensuring accurate repair rather than by removing damaged cells. The practical corollary is that any experiment has to be generation-controlled, and any strain has to be maintained against a frozen reference. |
| Recommended background | Standard N2 background, with an important husbandry constraint: because the phenotype is progressive, a line cannot simply be passaged indefinitely. The recommended practice for the reference allele is to cross once or twice, freeze many independent F2 plates, and return to those plates every couple of months for fresh stock, rather than continuously propagating one line. Telomere length and the generation number must be recorded alongside any phenotype. A BstNI restriction fragment length polymorphism linked to the reference mutation makes the allele straightforward to track through crosses. |
| Representative allele | The reference allele mrt-2(e2663), together with the deletion allele mrt-2(ok1260) (about 2600 bp removed). Custom CRISPR alleles on request: full deletions, point substitutions in the clamp interface or the PCNA-like fold, GFP / mCherry / 3xFLAG knockins at the endogenous locus, and conditional degron insertions. Because the phenotype accumulates over generations, we recommend establishing at least two independent edited lines and freezing them early rather than expanding a single isolate. |
mrt-2 at a glance: mrt-2 gives a progressive phenotype: fertile for ~F10-F28 then sterile, so freeze many F2 plates and track generation number. Editing mrt-2 alone is a powerful way to dissect MRT-2 function in vivo, with conserved relevance to human RAD1 biology.
gRNA-guided Cas9 cutting with NHEJ repair to introduce frameshift indels, generating premature stop codons and null / strong-loss-of-function mrt-2 alleles.
Homology-directed repair (HDR) inserts GFP / HA / FLAG or other tags at the mrt-2 locus for endogenous expression and subcellular localization studies.
Introduce specific amino-acid substitutions to dissect functional domains and separate catalytic from scaffolding roles of MRT-2.
Build tissue-specific, inducible, or temperature-sensitive configurations and reporter lines for spatiotemporal analysis of MRT-2 function.
The design issue here is not the edit but the timeline. Because sterility appears only after many generations, a fresh knockout looks superficially wild type, and a negative result scored at F2 is meaningless. Plan from the outset to carry at least two independent edited lines in parallel, record the generation number for every assay, and freeze multiple F2 plates as a permanent reference before the lines begin to decline. Measure telomere length directly rather than inferring it, since the initial length varies between strains through random segregation and the erosion rate is the meaningful quantity. If the project needs an acute, non-progressive readout of the same pathway, the apoptotic corpse count after irradiation is the faster endpoint, and cep-1 gives a clean comparator that lacks the genome-instability component entirely.
Define target (knockout / knockin / point mutation), select gRNA(s) and HDR template, confirm against the latest genome build.
Design, validate, and synthesize high-specificity guide RNAs targeting the mrt-2 locus.
Build Cas9 / gRNA delivery construct and, for knockin, the homology-arm repair template.
Perform gonadal microinjection of N2 animals with CRISPR components and co-CRISPR selection markers.
PCR, Sanger / sequencing, and phenotype confirmation to isolate edited animals and verify alleles.
Provide homozygous / balanced mrt-2 strains plus a full genotype and methods report.
Standard knockout projects deliver a frameshift indel allele predicted to produce a premature stop and a loss-of-function phenotype. We confirm the edit by sequencing and, where relevant, by characteristic phenotype assays.
Yes. Using HDR with a designed repair template, we can insert GFP, HA, or FLAG tags at the C- or N-terminus while preserving endogenous promoter and regulation.
Timelines vary with editing type and validation needs, but our optimized pipeline and co-CRISPR selection typically shorten turnaround versus conventional approaches. Contact us with your design for a project-specific estimate.
Because the phenotype is progressive and trans-generational rather than acute. The 9-1-1 clamp is required for telomerase to act at chromosome ends, so telomeres shorten by roughly 130 base pairs per generation — the same rate seen in trt-1 telomerase mutants, and not additive in the double mutant, which is the genetic evidence that the two act in one pathway. Lines therefore reproduce for a number of generations and then become sterile as telomeres erode and chromosomes fuse end to end; with the reference allele that window closes somewhere between generation F10 and F28. The high-incidence-of-males phenotype worsens in later generations as X-autosome fusions accumulate. Maintain the strain accordingly: cross once or twice, freeze many independent F2 plates, and go back to those plates every couple of months for fresh stock instead of continuously passaging one line. Record the generation number alongside every measurement. A BstNI polymorphism linked to the mutation makes it easy to follow through crosses. Note also the useful negative control: mutants lacking only the apoptotic branch, such as cep-1, ced-3 and ced-4, do not show the mutator phenotype, so genome instability and cell death are separable outcomes here.
CD BioSciences is committed to convenient, efficient, and cost-effective C. elegans model customization. Tell us your target design — knockout, knockin, tagging, or point mutation — and our scientists will respond with a tailored plan and quote.
Contact UsReferences
1. Kim H. M., Colaiácovo M. P. CRISPR-Cas9-guided genome engineering in C. elegans. Current Protocols in Molecular Biology, 2016, 115(1): 31.7.1–31.7.18.
2. Harris J., Lowden M., Clejan I., Tzoneva M., Thomas J.H., Hodgkin J., Ahmed S. Mutator phenotype of Caenorhabditis elegans DNA damage checkpoint mutants. Genetics, 2006, 174(2): 601–616.
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