Effects of faf1 gene knockout by CRISPR/Cas9 on zebrafish cartilage and sarcomere development
10.16016/j.1000-5404.201612182
- VernacularTitle:CRISPR/Cas9技术敲除faf1基因对斑马鱼软骨及肌节发育的影响
- Author:
Jing LIU
;
Sen LIANG
;
Zhi YUAN
;
Huizhe HUANG
- Keywords:
faf1;
zebrafish;
CRISPR/Cas9;
cartilage development;
delayed pigmentation;
sarcoidosis
- From:Journal of Third Military Medical University
2017;39(17):1709-1714
- CountryChina
- Language:Chinese
-
Abstract:
Objective To determine the effect of knocking down zebrafish faf1 gene by CRISPR/Cas9 editing technique.Methods gRNA was designed and prepared for the faf1 gene of zebrafish,and gRNA was mixed with Cas9 mRNA by microinjection into zebrafish single cell embryos.The mutant F0 generation zebrafish was screened out by enzyme digestion and gene sequencing.The mutant F0 was genetically outcrossed with the wild-type zebrafish to get the F1 heterozygous zebrafish,and the genotype of zebrafish was detected by microscopic observation.Results The faf1 gRNA and Cas9 mRNA were successfully prepared.The gRNA (gRNA6) located in the exon 6 of faf1 could shift the faf1 gene into frameshift mutations.The mutation type MU1 was screened out and the somatic cytochrome deposition delay was observed in this heterozygous zebrafish.At 4 d post fertilization (dpf),there were sarcomeric dysplasia and head shrinkage,increased hyoid angle and other craniofacial cartilage deformities.And the zebrafish died at 8 ～9 dpf.Conclusion CRISPR/Cas9 knocking out thefaf1 gene produces a new phenotype for zebrafish,with delayed pigment deposition and nodule-like change in tail muscle section.
