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CRISPR/Cas9 Genome Editing01:28

CRISPR/Cas9 Genome Editing

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The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids, forming the foundation for its adaptation as a powerful genome-editing tool. Originally discovered in prokaryotes, this system has been repurposed to revolutionize genetic engineering across a wide range of organisms, including plants, animals, and humans. The core component, Cas9, is an endonuclease derived from Streptococcus pyogenes, capable of introducing...
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Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced...
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Bacteria and archaea are susceptible to viral infections just like eukaryotes; therefore, they have developed a unique adaptive immune system to protect themselves. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) are present in more than 45% of known bacteria and 90% of known archaea.
The CRISPR-Cas system stores a copy of foreign DNA in the host genome and uses it to identify the foreign DNA upon reinfection. CRISPR-Cas has three different...
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Related Experiment Video

Updated: Mar 14, 2026

Screening Sperm for the Rapid Isolation of Germline Edits in Zebrafish
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Optimized CRISPR-Cas9 System for Genome Editing in Zebrafish.

Charles E Vejnar1, Miguel A Moreno-Mateos1, Daniel Cifuentes1

  • 1Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06510.

Cold Spring Harbor Protocols
|October 5, 2016
PubMed
Summary

This protocol optimizes CRISPR-Cas9 genome editing in zebrafish using CRISPRscan to design efficient single guide RNAs (sgRNAs). It details generating and genotyping mutants, enhancing viability by targeting germline Cas9, and identifying heterozygous fish.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Zebrafish Model Organisms

Background:

  • CRISPR-Cas9 is a powerful genome-editing tool, but sgRNA efficiency varies.
  • Optimizing sgRNA design is crucial for efficient and reliable gene editing in zebrafish.
  • Previous methods for generating and genotyping zebrafish mutants have limitations.

Purpose of the Study:

  • To describe an optimized CRISPR-Cas9 protocol for generating and genotyping zebrafish mutants.
  • To introduce and utilize the CRISPRscan web tool for designing highly efficient sgRNAs.
  • To present methods for increasing mutant viability and accurately identifying heterozygous F1 fish.

Main Methods:

  • Design of highly efficient single guide RNAs (sgRNAs) using the CRISPRscan algorithm, based on analysis of over 1000 zebrafish sgRNAs.
  • In vitro generation of sgRNAs for in vivo injection to target specific genomic loci.
  • Germline targeting of Cas9 to enhance embryo viability by reducing somatic mutations.
  • Identification of F1 heterozygous fish using Mut-Seq (high-throughput sequencing for F0 founders) and polymerase chain reaction-based fragment analysis.

Main Results:

  • The CRISPRscan tool identifies highly predictive factors for Cas9 activity, enabling the design of efficient sgRNAs.
  • Targeting Cas9 to the germline significantly increases the viability of mutant zebrafish embryos.
  • A combined approach of Mut-Seq and fragment analysis accurately identifies F1 heterozygous fish carrying desired mutations.

Conclusions:

  • This protocol provides a robust and optimized method for generating and characterizing mutant zebrafish lines using CRISPR-Cas9.
  • The CRISPRscan tool and described genotyping methods streamline the process of creating genetically modified zebrafish.
  • The protocol facilitates efficient creation of zebrafish models for genetic research.