[Complexity of Detecting CRISPR/Cas9-Mediated Homologous Recombination in Zebrafish]

Area of Science:

• Genetics
• Molecular Biology
• Developmental Biology

Background:

• Homology-directed (HD) genome modification enables precise genetic alterations.
• In vivo genome editing efficiency varies significantly across different genomic loci.
• CRISPR/Cas9 technology offers potential for targeted gene knock-ins.

Purpose of the Study:

• To investigate the precise knock-in of a GFP reporter into the gad locus in zebrafish (Danio rerio) using CRISPR/Cas9.
• To identify and address challenges related to homology-directed repair (HDR) efficiency in vivo.
• To understand factors contributing to the loss of targeted insertion events.

Main Methods:

• CRISPR/Cas9 system employed for targeted gene editing.
• Homologous recombination (HR) assessed via PCR and GFP reporter expression.
• Optimization of PCR conditions and targeting plasmid concentration to mitigate artifacts.
• Time-course analysis of HR-positive embryos and GFP signal during embryogenesis.

Main Results:

• PCR artifacts indicative of homologous recombination were initially detected but subsequently mitigated.
• A decline in HR-positive embryos and diminishing GFP signals were observed as embryogenesis progressed.
• GFP signal intensity correlated with PCR detection, suggesting loss of targeted insertions over time.
• Loss of targeted insertion events was identified as a potential reason for failed germline transmission.

Conclusions:

• CRISPR-mediated knock-in efficiency is impacted by the loss of targeted integration events post-insertion.
• The instability of insertions at the gad locus in zebrafish contributes to low overall HR efficiency.
• Understanding and overcoming post-integration loss is crucial for successful germline editing in zebrafish.