[A comparison of the knockout efficiencies of two codon-optimized Cas9 coding sequences in zebrafish embryos]

Area of Science:

• Molecular Biology
• Genetics
• Zebrafish Models

Background:

• The CRISPR/Cas9 system has rapidly advanced for gene editing.
• Efficient and specific gene knockout in zebrafish is crucial for research.
• Different labs have developed distinct Cas9 cDNA sequences for zebrafish.

Purpose of the Study:

• To compare the gene knockout efficiency of two zebrafish-codon-optimized Cas9 sequences (zCas9_bz and zCas9_wc).
• To evaluate these sequences for knocking out seven genes (exogenous egfp and endogenous chd, hbegfa, th, eef1a1b, tyr, tcf7l1a) in zebrafish embryos.

Main Methods:

• Utilized two distinct zebrafish-codon-optimized Cas9 coding sequences (zCas9_bz, zCas9_wc).
• Applied these sequences for gene knockout in zebrafish embryos targeting seven genes.
• Assessed knockout efficiency through direct sequencing of PCR products, colony sequencing, and phenotypic analysis.

Main Results:

• The zCas9_wc sequence consistently showed higher gene knockout efficiency compared to the zCas9_bz sequence.
• This higher efficiency was observed across all tested genes and conditions.

Conclusions:

• The zCas9_wc zebrafish-codon-optimized Cas9 sequence is more effective for gene knockout in zebrafish embryos than zCas9_bz.
• This finding provides valuable information for selecting optimal CRISPR/Cas9 components for zebrafish gene editing studies.