Improving CRISPR/Cas9 mutagenesis efficiency by delaying the early development of zebrafish embryos

CRISPR/Cas9 driven mutagenesis in zygotes is a popular tool for introducing targeted mutations in model organisms. Compared to mouse, mutagenesis in zebrafish is relatively inefficient and results in somatic mosaicism most likely due to a short single-cell stage of about 40 min. Here we explored two...

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Bibliographic Details
Published in:Scientific reports 2020-12, Vol.10 (1), p.21023, Article 21023
Main Authors: Terzioglu, M., Saralahti, A., Piippo, H., Rämet, M., Andressoo, J.-O.
Format: Article
Language:English
Subjects:
631/1647/1513/1967/3196
631/1647/334/1874/763
Animals
Cell division
CRISPR
CRISPR-Cas Systems
Danio rerio
Developmental stages
Embryos
Female
Follicular Fluid - metabolism
Gene Editing - methods
Humanities and Social Sciences
Hydrogen sulfide
Hydrogen Sulfide - pharmacology
In vitro fertilization
Mosaicism
multidisciplinary
Mutagenesis
Oncorhynchus mykiss
Oncorhynchus tshawytscha
Oocytes
Oocytes - drug effects
Oocytes - metabolism
Ovaries
Salmon
Science
Science (multidisciplinary)
Temperature
Zebrafish - genetics
Zebrafish - growth & development
Zygote - cytology
Zygote - metabolism
Zygotes
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Summary:CRISPR/Cas9 driven mutagenesis in zygotes is a popular tool for introducing targeted mutations in model organisms. Compared to mouse, mutagenesis in zebrafish is relatively inefficient and results in somatic mosaicism most likely due to a short single-cell stage of about 40 min. Here we explored two options to improve CRISPR/Cas9 mutagenesis in zebrafish—extending the single-cell stage and defining conditions for carrying out mutagenesis in oocytes prior to in vitro fertilization. Previous work has shown that ovarian fluid from North American salmon species (coho and chinook salmon) prolong oocyte survival ex vivo so that they are viable for hours instead of dying within minutes if left untreated. We found that commonly farmed rainbow trout ( Oncorhynchus mykiss ) ovarian fluid (RTOF) has similar effect on zebrafish oocyte viability. In order to prolong single-cell stage, we incubated zebrafish zygotes in hydrogen sulfide (H 2 S) and RTOF but failed to see any effect. However, the reduction of temperature from standard 28 to 12 °C postponed the first cell division by about an hour. In addition, the reduction in temperature was associated with increased CRISPR/Cas9 mutagenesis rate. These results suggest that the easily applicable reduction in temperature facilitates CRISPR/Cas9 mutagenesis in zebrafish.
ISSN:2045-2322
2045-2322
DOI:10.1038/s41598-020-77677-9