Optimized knock-in of point mutations in zebrafish using CRISPR/Cas9

Area of Science:

• Molecular Biology
• Genetics
• Zebrafish Models

Background:

• Precise genetic modifications are crucial for disease modeling.
• CRISPR/Cas9 technology offers powerful gene editing capabilities.
• Optimizing knock-in efficiency in zebrafish remains a challenge.

Purpose of the Study:

• To optimize point mutation knock-ins in zebrafish using CRISPR/Cas9.
• To develop reliable methods for assessing knock-in efficiency and accuracy.
• To identify strategies for improving knock-in success rates.

Main Methods:

• Utilized clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 reagents and single-stranded oligodeoxynucleotides for knock-ins.
• Employed allele-specific polymerase chain reaction and high-throughput sequencing for efficiency assessment.
• Investigated various oligo design and modification strategies.

Main Results:

• Anti-sense asymmetric oligo design proved most successful.
• Cut site proximity to the mutation and phosphorothioate modifications significantly improved efficiency.
• Identified and mitigated off-target trans knock-ins through a novel detection workflow.

Conclusions:

• Optimized CRISPR/Cas9 strategies enhance point mutation knock-in efficiency in zebrafish.
• Developed methods for accurate knock-in detection, addressing off-target risks.
• These advancements facilitate genetic disease research in zebrafish and other animal models.