Non-coding RNAs mediate the rearrangements of genomic DNA in ciliates

Feng, Xuezhu; Guang, Shouhong

doi:10.1007/s11427-013-4539-4

Cited by 4 publications

(2 citation statements)

References 66 publications

(75 reference statements)

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“…RNA interference and the creation of deletion mutants have both long been used to perturb gene function in C. elegans . The low efficacy and off-target effects of RNAi require null mutants that eliminate gene functions under many circumstances 42 43 47 . To generate knockout mutants in C. elegans , the random mutagen trimethylpsoralen (TMP) followed by UV irradiation was used to mutagenize a very large number of worms followed by PCR screening 48 49 50 .…”

Section: Discussionmentioning

confidence: 99%

Dual sgRNA-directed gene knockout using CRISPR/Cas9 technology in Caenorhabditis elegans

Chen

Zhu

et al. 2014

Sci Rep

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The CRISPR RNA-guided Cas9 nuclease gene-targeting system has been successfully used for genome editing in a variety of organisms. Here, we report the use of dual sgRNA-guided Cas9 nuclease to generate knockout mutants of protein coding genes, noncoding genes, and repetitive sequences in C. elegans. Co-injection of C. elegans with dual sgRNAs results in the removal of the interval between two sgRNAs and the loss-of-function phenotype of targeted genes. We sought to determine how large an interval can be eliminated and found that at least a 24 kb chromosome segment can be deleted using this dual sgRNA/Cas9 strategy. The deletion of large chromosome segments facilitates mutant screening by PCR and agarose electrophoresis. Thus, the use of the CRISPR/Cas9 system in combination with dual sgRNAs provides a powerful platform with which to easily generate gene knockout mutants in C. elegans. Our data also suggest that encoding multiple sgRNA sequences into a single CRISPR array to simultaneously edit several sites within the genome may cause the off-target deletion of chromosome sequences.

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Section: Discussionmentioning

confidence: 99%

Dual sgRNA-directed gene knockout using CRISPR/Cas9 technology in Caenorhabditis elegans

Chen

Zhu

et al. 2014

Sci Rep

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126

131

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“…Although there are numerous copies of rDNA in ciliate macronucleus, it is likely that only a small portion of these genes are transcriptionally active in ciliates under any given growth condition, as previously shown for other eukaryotes ( Reeder, 1999 ). Studies in Tetrahymena thermophila , Paramecium tetraurelia , and Oxytricha trifallax have pinpointed the important role of non-coding RNAs in genome rearrangement events ( Feng and Guang, 2013 ). In spirotrichous ciliates, such as Euplotes , Stylonychia , and Oxytricha , more than 95% of the micronuclear DNA is eliminated to form the macronucleus during sexual reproduction ( Swart et al, 2013 ) possible as a mechanism to allow the cell to adapt during times of stress.…”

Section: Functional Genes In Rumen Ciliate Protozoamentioning

confidence: 99%

The Role of Ciliate Protozoa in the Rumen

et al. 2015

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First described in 1843, Rumen protozoa with their striking appearance were assumed to be important for the welfare of their host. However, despite contributing up to 50% of the bio-mass in the rumen, the role of protozoa in rumen microbial ecosystem remains unclear. Phylogenetic analysis of 18S rDNA libraries generated from the rumen of cattle, sheep, and goats has revealed an unexpected diversity of ciliated protozoa although variation in gene copy number between species makes it difficult to obtain absolute quantification. Despite repeated attempts it has proven impossible to maintain rumen protozoa in axenic culture. Thus it has been difficult to establish conclusively a role of ciliate protozoa in rumen fiber degradation. The development of techniques to clone and express ciliate genes in λ phage, together with bioinformatic indices to confirm the ciliate origin of the genes has allowed the isolation and characterization of fibrolytic genes from rumen protozoa. Elimination of the ciliate protozoa increases microbial protein supply by up to 30% and reduces methane production by up to 11%. Our recent findings suggest that holotrich protozoa play a disproportionate role in supporting methanogenesis whilst the small Entodinium are responsible for much of the bacterial protein turnover. As yet no method to control protozoa in the rumen that is safe and practically applicable has been developed, however a range of plant extract capable of controlling if not completely eliminating rumen protozoa have been described.

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