ZFIN ID: ZDB-PUB-210123-25
An improved method for precise genome editing in zebrafish using CRISPR-Cas9 technique
Gasanov, E.V., Jędrychowska, J., Pastor, M., Wiweger, M., Methner, A., Korzh, V.P.
Date: 2021
Source: Molecular biology reports   48(2): 1951-1957 (Other)
Registered Authors: Jędrychowska, Justyna, Korzh, Vladimir, Wiweger, Malgorzata
Keywords: CRISPR-Cas9, HRM, Precise deletion editing, Zebrafish, gRNAs
MeSH Terms:
  • Animals
  • CRISPR-Cas Systems*
  • Gene Deletion
  • Gene Editing/methods*
  • Heterozygote
  • Nerve Tissue Proteins/genetics
  • Nucleic Acid Denaturation
  • RNA, Guide/genetics*
  • Voltage-Dependent Anion Channels/genetics
  • Zebrafish/genetics*
  • Zebrafish Proteins/genetics
PubMed: 33481178 Full text @ Mol. Biol. Rep.
Current methods of CRISPR-Cas9-mediated site-specific mutagenesis create deletions and small insertions at the target site which are repaired by imprecise non-homologous end-joining. Targeting of the Cas9 nuclease relies on a short guide RNA (gRNA) corresponding to the genome sequence approximately at the intended site of intervention. We here propose an improved version of CRISPR-Cas9 genome editing that relies on two complementary guide RNAs instead of one. Two guide RNAs delimit the intervention site and allow the precise deletion of several nucleotides at the target site. As proof of concept, we generated heterozygous deletion mutants of the kcng4b, gdap1, and ghitm genes in the zebrafish Danio rerio using this method. A further analysis by high-resolution DNA melting demonstrated a high efficiency and a low background of unpredicted mutations. The use of two complementary gRNAs improves CRISPR-Cas9 specificity and allows the creation of predictable and precise mutations in the genome of D. rerio.