Genome editing using CRISPR/Cas9-based knock-in approaches in zebrafish

Area of Science:

• Molecular Biology
• Genetics
• Zebrafish Model Organisms

Background:

• CRISPR/Cas9 genome editing technology is rapidly advancing, with increasing reports of knock-out studies in zebrafish.
• Generating knock-in models in zebrafish using CRISPR/Cas9 has been challenging, particularly with homologous directed repair (HDR) methods.

Purpose of the Study:

• To provide an overview of homologous directed repair (HDR)-based knock-in generation in zebrafish.
• To discuss limitations of HDR and explore alternative non-homologous dependent strategies for knock-in generation.

Main Methods:

• Review of existing literature on CRISPR/Cas9-mediated knock-in strategies in zebrafish.
• Analysis of critical factors influencing HDR efficiency, including single guide RNA design, Cas9 delivery, and homology arm length.
• Examination of non-homologous end-joining (NHEJ)-based strategies for generating knock-ins.

Main Results:

• Homologous directed repair (HDR) in zebrafish for knock-ins faces several critical limitations affecting efficiency and insertion rates.
• Non-homologous end-joining (NHEJ)-based strategies, while less precise, demonstrate higher efficiency and insertion rates for generating reporter lines or editing open reading frames.
• NHEJ-based knock-ins are presented as a promising approach to overcome HDR limitations for targeting endogenous loci in zebrafish.

Conclusions:

• Non-homologous end-joining (NHEJ) offers a more efficient and versatile alternative to HDR for CRISPR/Cas9-based knock-ins in zebrafish.
• Proposed new strategies for cDNA edited or tagged insertions using NHEJ will enhance the CRISPR/Cas9 toolbox for zebrafish research.