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Targeted RNA knockdown by crRNA guided Csm in zebrafish

Thomas Fricke, Gintautas Tamulaitis, Dalia Smalakyte, Michal Pastor, Agnieszka Kolano, Virginijus Siksnys, View ORCID ProfileMatthias Bochtler
doi: https://doi.org/10.1101/228775
Thomas Fricke
1International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw, Poland
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Gintautas Tamulaitis
2Institute of Biotechnology, Vilnius University, Saulėtekio av. 7, 10257 Vilnius, Lithuania
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Dalia Smalakyte
2Institute of Biotechnology, Vilnius University, Saulėtekio av. 7, 10257 Vilnius, Lithuania
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Michal Pastor
3Polish Academy of Sciences, Institute of Biochemistry and Biophysics, Pawinskiego 5a, 02-106 Warsaw, Poland
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Agnieszka Kolano
1International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw, Poland
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Virginijus Siksnys
2Institute of Biotechnology, Vilnius University, Saulėtekio av. 7, 10257 Vilnius, Lithuania
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  • For correspondence: siksnys@ibt.lt mbochtler@iimcb.gov.pl
Matthias Bochtler
1International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw, Poland
3Polish Academy of Sciences, Institute of Biochemistry and Biophysics, Pawinskiego 5a, 02-106 Warsaw, Poland
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  • ORCID record for Matthias Bochtler
  • For correspondence: siksnys@ibt.lt mbochtler@iimcb.gov.pl
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ABSTRACT

RNA interference (RNAi) is a powerful experimental tool for RNA knockdown, but not all organisms are amenable. Here, we provide a “proof of principle” demonstration that CRISPR endoribonucleases can be used for programmable mRNA transcript degradation. Using zebrafish as the animal model and Csm(crRNA) complexes as the CRISPR endoribonucleases, we have targeted a transgenic EGFP transcript expressed from a variety of promoters. A drastic decrease of fluorescence was achieved in germ cells of the vasa:EGFP line. Weaker effects were also seen in fish lines that express EGFP zygotically. Knockdown was statistically significant in cmcl2:EGFP and fli1:EGFP zebrafish lines at 1 day post fertilization (dpf), but reduced to background levels at 2 dpf. The nkx2.5:EGFP fish line was least susceptible to Csm mediated EGFP knockdown. We conclude that at the present stage, Csm mediated knockdown is already efficient for maternal transcripts, and may compare favorably with morpholinos for such targets in zebrafish.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-ND 4.0 International license.
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Posted December 04, 2017.
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Targeted RNA knockdown by crRNA guided Csm in zebrafish
Thomas Fricke, Gintautas Tamulaitis, Dalia Smalakyte, Michal Pastor, Agnieszka Kolano, Virginijus Siksnys, Matthias Bochtler
bioRxiv 228775; doi: https://doi.org/10.1101/228775
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Targeted RNA knockdown by crRNA guided Csm in zebrafish
Thomas Fricke, Gintautas Tamulaitis, Dalia Smalakyte, Michal Pastor, Agnieszka Kolano, Virginijus Siksnys, Matthias Bochtler
bioRxiv 228775; doi: https://doi.org/10.1101/228775

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