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ZFIN ID: ZDB-PUB-160226-17
A comparison of the knockout efficiencies of two codon-optimized Cas9 coding sequences in zebrafish embryos
Fenghua, Z., Houpeng, W., Siyu, H., Feng, X., Zuoyan, Z., Yonghua, S.
Date: 2016
Source: Yi chuan = Hereditas   38: 144-54 (Journal)
Registered Authors: Zhu, Zuoyan
Keywords: CRISPR/Cas9, zebrafish, gene knockout, mutation efficiency
MeSH Terms:
  • Amino Acid Sequence
  • Animals
  • Animals, Genetically Modified
  • Base Sequence
  • CRISPR-Cas Systems*
  • Codon/genetics*
  • Embryo, Nonmammalian/embryology
  • Embryo, Nonmammalian/metabolism*
  • Female
  • Gene Expression Regulation, Developmental
  • Green Fluorescent Proteins/genetics
  • Green Fluorescent Proteins/metabolism
  • Male
  • Molecular Sequence Data
  • RNA, Messenger/genetics
  • RNA, Messenger/metabolism
  • Sequence Homology, Amino Acid
  • Time Factors
  • Zebrafish/embryology
  • Zebrafish/genetics*
  • Zebrafish Proteins/genetics*
  • Zebrafish Proteins/metabolism
PubMed: 26907778 Full text @ Yi Chuan
Recent years have witnessed the rapid development of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein(CRISPR/Cas9)system. In order to realize gene knockout with high efficiency and specificity in zebrafish, several labs have synthesized distinct Cas9 cDNA sequences which were cloned into different vectors. In this study, we chose two commonly used zebrafish-codon-optimized Cas9 coding sequences (zCas9_bz, zCas9_wc) from two different labs, and utilized them to knockout seven genes in zebrafish embryos, including the exogenous egfp and six endogenous genes (chd, hbegfa, th, eef1a1b, tyr and tcf7l1a). We compared the knockout efficiencies resulting from the two zCas9 coding sequences, by direct sequencing of PCR products, colony sequencing and phenotypic analysis. The results showed that the knockout efficiency of zCas9_wc was higher than that of zCas9_bz in all conditions.
Article in Chinese.