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Related Concept Videos

In-vitro Mutagenesis01:16

In-vitro Mutagenesis

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To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.
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Related Experiment Video

Updated: Jul 17, 2025

CRISPR-Cas9-Mediated Precise Knock-In Edits in Zebrafish Hearts
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Mutation Knock-in Methods Using Single-Stranded DNA and Gene Editing Tools in Zebrafish.

Sergey V Prykhozhij1, Jason N Berman2,3

  • 1Children's Hospital of Eastern Ontario Research Institute, Ottawa, ON, Canada.

Methods in Molecular Biology (Clifton, N.J.)
|September 5, 2023
PubMed
Summary

CRISPR/Cas9 gene editing in zebrafish is enhanced using single-stranded DNA (ssDNA) templates for precise genomic modifications. This review details ssDNA methods, challenges, and solutions for generating mutant zebrafish lines.

Keywords:
CRISPR/Cas9Disease modelKnock-inLong ssDNAPoint mutationZebrafishssODN

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Area of Science:

  • Genetics and Genomics
  • Molecular Biology
  • Zebrafish Model Systems

Background:

  • CRISPR/Cas9 technology enables precise genomic modifications, crucial for model systems like zebrafish.
  • Single-stranded DNA (ssDNA) is the primary donor template for knock-in applications, available as ssODNs or lssDNA.
  • While base and prime editing are emerging, ssDNA-based methods offer broader applicability for complex knock-ins.

Approach:

  • This review examines CRISPR/Cas9 knock-in strategies in zebrafish utilizing ssDNA donor templates.
  • It highlights methods to enhance knock-in efficiency, including lssDNA and tethering strategies.
  • Potential challenges in DNA repair and knock-in are discussed, with proposed solutions.

Key Points:

  • ssDNA donor templates (ssODN and lssDNA) are effective for precise CRISPR/Cas9 knock-ins in zebrafish.
  • Optimizing donor template design and delivery enhances knock-in efficiency.
  • Complementarity exists between ssDNA-based methods and newer editing techniques like base and prime editing.

Conclusions:

  • ssDNA remains a versatile and powerful tool for generating precise zebrafish mutants via CRISPR/Cas9.
  • Understanding and overcoming DNA repair challenges are key to successful knock-in.
  • A combination of editing technologies will likely be employed for comprehensive mutant generation.