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.
Subject(s)
CRISPR-Cas Systems , Gene Editing/methods , Mutagenesis , Zebrafish/genetics , Zygote/metabolism , Animals , Female , Follicular Fluid/metabolism , Hydrogen Sulfide/pharmacology , Oocytes/drug effects , Oocytes/metabolism , Temperature , Zebrafish/growth & development , Zygote/cytologySubject(s)
Glucose-6-Phosphatase/genetics , Glycogen Storage Disease Type I/enzymology , Glycogen Storage Disease Type I/genetics , Mutation , Alleles , Child, Preschool , Consanguinity , Gene Frequency , Genotype , Glycogen Storage Disease Type I/diagnosis , Homozygote , Humans , Infant , Liver/enzymology , Phenotype , TurkeyABSTRACT
Pseudodeficiency (Pd) in arylsulfatase A (ASA) is a relatively frequent condition in healthy individuals. It produces a reduction in enzyme activity similar to that found in metachromatic leukodystrophy (MLD). A variable incidence of the Pd allele was found in different populations; it was 10-20 times higher than that of metachromatic leukodystrophy. Twelve of the 52 unrelated, healthy individuals were found to be heterozygous for the ASA Pd allele. In Turkey we estimated the incidence of the Pd allele as 11.5 percent. Out of 18 cases with MLD, one patient was found homozygous for the Pd allele and the other patient was found heterozygous.
