Targeted Chromosomal Rearrangements via Combinatorial Use of CRISPR/Cas9 and Cre/<i>LoxP</i>Technologies in<i>Caenorhabditis elegans</i>

Chen, Xiangyang; Liao, Shimiao; Huang, Xinya; Xu, Ting; Feng, Xuezhu; Guang, Shouhong

doi:10.1534/g3.118.200473

Cited by 11 publications

(15 citation statements)

References 46 publications

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“…These four fragments were joined together by Gibson assembly to form the rbd-1::mCherry repair plasmid. The transgene was integrated onto the C. elegans ' chromosome I via a modified counterselection (cs)-CRISPR method ( 23 ).…”

Section: Methodsmentioning

confidence: 99%

Antisense ribosomal siRNAs inhibit RNA polymerase I-directed transcription in C. elegans

Liao

Chen

et al. 2021

Nucleic Acids Research

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Eukaryotic cells express a wide variety of endogenous small regulatory RNAs that function in the nucleus. We previously found that erroneous rRNAs induce the generation of antisense ribosomal siRNAs (risiRNAs) which silence the expression of rRNAs via the nuclear RNAi defective (Nrde) pathway. To further understand the biological roles and mechanisms of this class of small regulatory RNAs, we conducted forward genetic screening to identify factors involved in risiRNA generation in Caenorhabditis elegans. We found that risiRNAs accumulated in the RNA exosome mutants. risiRNAs directed the association of NRDE proteins with pre-rRNAs and the silencing of pre-rRNAs. In the presence of risiRNAs, NRDE-2 accumulated in the nucleolus and colocalized with RNA polymerase I. risiRNAs inhibited the transcription elongation of RNA polymerase I by decreasing RNAP I occupancy downstream of the RNAi-targeted site. Meanwhile, exosomes mislocalized from the nucleolus to nucleoplasm in suppressor of siRNA (susi) mutants, in which erroneous rRNAs accumulated. These results established a novel model of rRNA surveillance by combining ribonuclease-mediated RNA degradation with small RNA-directed nucleolar RNAi system.

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Section: Methodsmentioning

confidence: 99%

Antisense ribosomal siRNAs inhibit RNA polymerase I-directed transcription in C. elegans

Liao

Chen

et al. 2021

Nucleic Acids Research

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“…These five fragments were joined together by Gibson assembly to form the susi-2::mCherry repair plasmid. The transgene was integrated onto the C. elegans ’ chromosome II via a modified counterselection (cs)-CRISPR method [45].…”

Section: Methodsmentioning

confidence: 99%

“…For in situ transgene 3xflag::gfp::rpoa-2, the 3xFLAG::GFP coding region was PCR amplified from YY174 genomic DNA with the primers 5′- The transgene was integrated onto the C. elegans' chromosome II via a modified counterselection (cs)-CRISPR method [45].…”

Section: Construction Of Plasmids and Transgenic Strainsmentioning

confidence: 99%

Co-surveillance of ribosomal RNA by the exosome complex and nucleolar RNAi inC. elegans

Liao

Chen

et al. 2020

Preprint

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Eukaryotic cells express a wide variety of endogenous small regulatory RNAs that function in the nucleus. We previously found that erroneous rRNAs induce the generation of antisense ribosomal siRNAs (risiRNAs) which silence the expression of rRNAs via the nuclear RNAi defective (Nrde) pathway. To further understand the biological roles and mechanisms of this class of small regulatory RNAs, we conducted forward genetic screening to identify factors involved in risiRNA generation in Caenorhabditis elegans. We found that risiRNAs accumulated in the RNA exosome mutants. risiRNAs directed a NRDE-dependent silencing of pre-rRNAs in the nucleolus. In the presence of risiRNA, NRDE-2 accumulated in the nucleolus and colocalized with RNA polymerase I. risiRNA inhibited the transcription elongation of RNA polymerase I by decreasing RNAP I occupancy downstream of the site of RNAi. Meanwhile, exosome mislocalized from the nucleolus to nucleoplasm in suppressor of siRNA (susi) mutants, in which erroneous rRNAs accumulated. These results establish a novel model of rRNA surveillance by combining ribonuclease-mediated RNA degradation with small RNA-directed nucleolar RNAi system.

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“…Previous reports indicate that multiple CRISPR-mediated DNA doublestrand breaks at homologous chromosomal loci can lead to chromosomal aberrations such as inversions, large deletions, circularizations, and chromosomal cleavages [39][40][41][42][43][44][45] . To create strains with fragmented X chromosomes, we searched for genomic regions near the ends of chromosome X with homology to regions at the center.…”

Section: Creation Of C Elegans Stable Homozygous Strains With Brokenmentioning

confidence: 99%

Chromosome Integrity is Required for the Initiation of Meiotic Sex Chromosome Inactivation inCaenorhabditis elegans

Rappaport

Achache

Falk

et al. 2020

Preprint

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During meiosis of heterogametic cells, such as XY meiocytes, sex chromosomes of many species undergo transcriptional silencing known as meiotic sex chromosome inactivation (MSCI). Silencing also occurs in aberrantly unsynapsed autosomal chromatin. The silencing of unsynapsed chromatin, is assumed to be the underline mechanism for MSCI. Initiation of MSCI is disrupted in meiocytes with sex chromosome-autosome translocations. Whether this is due to aberrant synapsis or the lack of sex chromosome integrity has never been determined. To address this, we used CRISPR to engineer Caenorhabditis elegans stable strains with broken X chromosomes that didn't undergo translocations with autosomes. In early meiotic nuclei of these mutants, the X fragments lack silent chromatin modifications and instead the fragments are enriched with transcribing chromatin modifications. Moreover, the level of active RNA polymerase II staining on the X fragments in mutant nuclei is similar to that on autosomes, indicating active transcription on the X. Contrary to previous models, which predicted that any unsynapsed chromatin is silenced during meiosis, X fragments that did not synapse were robustly stained with RNA polymerase II and gene expression levels were high throughout the broken X. Therefore, lack of synapsis does not trigger MSCI if sex chromosome integrity is lost. Moreover, our results suggest that a unique character of the chromatin of sex chromosomes underlies their lack of meiotic silencing due to both unsynapsed chromatin and sex chromosome mechanisms when their integrity is lost.

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Targeted Chromosomal Rearrangements via Combinatorial Use of CRISPR/Cas9 and Cre/LoxPTechnologies inCaenorhabditis elegans

Cited by 11 publications

References 46 publications

Antisense ribosomal siRNAs inhibit RNA polymerase I-directed transcription in C. elegans

Antisense ribosomal siRNAs inhibit RNA polymerase I-directed transcription in C. elegans

Co-surveillance of ribosomal RNA by the exosome complex and nucleolar RNAi inC. elegans

Chromosome Integrity is Required for the Initiation of Meiotic Sex Chromosome Inactivation inCaenorhabditis elegans

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