PUBLICATION

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

Authors
Terzioglu, M., Saralahti, A., Piippo, H., Rämet, M., Andressoo, J.O.
ID
ZDB-PUB-201208-23
Date
2020
Source
Scientific Reports   10: 21023 (Journal)
Registered Authors
Rämet, Mika, Saralahti, Anni
Keywords
none
MeSH Terms
  • Animals
  • CRISPR-Cas Systems*
  • Female
  • Follicular Fluid/metabolism
  • Gene Editing/methods*
  • Hydrogen Sulfide/pharmacology
  • Mutagenesis*
  • Oocytes/drug effects
  • Oocytes/metabolism
  • Temperature
  • Zebrafish/genetics*
  • Zebrafish/growth & development
  • Zygote/cytology
  • Zygote/metabolism*
PubMed
33273577 Full text @ Sci. Rep.
Abstract
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 (H2S) 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.
Genes / Markers
Figures
Show all Figures
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping