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Related Concept Videos

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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CRISPR01:59

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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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Homologous Recombination02:31

Homologous Recombination

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The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
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CRISPR and crRNAs02:53

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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.
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Conservative Site-specific Recombination and Phase Variation02:53

Conservative Site-specific Recombination and Phase Variation

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Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
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Related Experiment Video

Updated: Oct 3, 2025

Construction of Homozygous Mutants of Migratory Locust Using CRISPR/Cas9 Technology
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A high-efficiency and versatile CRISPR/Cas9-mediated HDR-based biallelic editing system.

Xinyi Li1,2, Bing Sun1, Hongrun Qian1

  • 1College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China.

Journal of Zhejiang University. Science. B
|February 21, 2022
PubMed
Summary

This study introduces a new CRISPR/Cas9 system for efficient biallelic gene editing, overcoming challenges in creating homozygous cell lines for disease modeling. The method achieves high biallelic editing efficiency for accurate gene modification.

Keywords:
Biallelic editingCRISPR/Cas9Homology-directed repair (HDR)Homozygote

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • CRISPR/Cas9 is a powerful genome editing tool, but achieving accurate biallelic editing for homozygous cell line generation remains challenging.
  • Current methods often result in only monoallelic editing, hindering the establishment of consistent phenotypes for gene function studies and disease modeling.
  • There is a critical need for an efficient system to perform biallelic editing using CRISPR/Cas9.

Purpose of the Study:

  • To develop and validate an efficient CRISPR/Cas9-based system for biallelic genome editing.
  • To improve the generation of homozygous cell lines and animal models for studying gene function and diseases.
  • To establish a robust method for accurate knock-in via homology-directed repair (HDR) at both alleles.

Main Methods:

  • Developed three integrated selection systems, each containing drug-screening and fluorescent marker genes.
  • Utilized CRISPR/Cas9-induced homology-directed repair (HDR) to integrate selection cassettes flanked by homologous arms into target loci.
  • Enriched positively targeted cell clones using drug resistance screening and fluorescent microscopy.

Main Results:

  • Successfully applied the novel method to the amyloid precursor protein (APP) and presenilin 1 (PSEN1) loci.
  • Achieved up to 82.0% biallelic editing efficiency after optimization, demonstrating the system's effectiveness.
  • Validated the enrichment strategy for identifying successfully edited cell clones.

Conclusions:

  • The developed strategy offers a new and efficient approach for biallelic editing using CRISPR/Cas9.
  • This method facilitates the creation of homozygous cell lines and animal models, crucial for consistent phenotypic analysis.
  • The system lays the foundation for establishing more accessible and efficient disease models.