CRISPR/Cas9-mediated precise genome modification by a long ssDNA template in zebrafish

Area of Science:

• Genomics
• Molecular Biology
• Zebrafish Model Organisms

Background:

• Homology-directed repair (HDR) enables precise genome editing but suffers from low efficiency in many model organisms, including zebrafish.
• Long single-stranded DNA (lssDNA) molecules have emerged as effective donor templates for HDR, particularly in mice.
• Developing efficient HDR methods is crucial for advancing genetic research and disease modeling in zebrafish.

Purpose of the Study:

• To introduce and validate zLOST (zebrafish long single-stranded DNA template), a novel method for efficient and precise genome editing in zebrafish.
• To assess the efficiency of zLOST in mediating homology-directed repair (HDR) for gene knock-in and mutation generation.
• To demonstrate the utility of zLOST for modeling human diseases and achieving germline transmission in zebrafish.

Main Methods:

• Utilized long single-stranded DNA (lssDNA) templates for homology-directed repair (HDR) in zebrafish embryos.
• Assessed knock-in efficiency via phenotypic rescue of pigmentation at the tyrosinase (tyr) locus.
• Quantified zLOST knock-in efficiency at multiple loci (rps14, nop56, th) using next-generation sequencing.
• Modeled specific human disease mutations in zebrafish using the zLOST method.

Main Results:

• zLOST successfully restored pigmentation in 98% of albino zebrafish embryos, confirming precise HDR-mediated repair.
• Next-generation sequencing demonstrated clear improvements in knock-in efficiency at the rps14, nop56, and th loci.
• The zLOST method enabled precise modeling of human disease mutations in zebrafish.
• Achieved a significant germline transmission rate of up to 31.8% using the zLOST method.

Conclusions:

• zLOST is a highly effective method for zebrafish genome editing, significantly improving targeted DNA knock-in efficiency through HDR.
• This technique offers a valuable tool for generating precise mutations and modeling diseases in zebrafish.
• The high efficiency and germline transmission rates make zLOST a promising approach for genetic research in zebrafish.