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Related Concept Videos

CRISPR01:59

CRISPR

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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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CRISPR/Cas9 Genome Editing01:28

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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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Related Experiment Video

Updated: Feb 25, 2026

Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format
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A refined method to study gene dosage changes in vitro using CRISPR/Cas9.

Teresa P Raposo1,2, Henry O Ebili1,2,3, Mohammad Ilyas1,2

  • 1Division of Cancer and Stem Cells, Faculty of Medicine and Health Sciences, University of Nottingham, UK.

Journal of Clinical Pathology
|August 11, 2017
PubMed
Summary

We developed an efficient CRISPR/Cas9 method to create gene dosage models in vitro. This technique uses Cas9 mRNA and labeled guide RNA for rapid mutation screening, enabling new studies of gene function.

Keywords:
Cancer GeneticsMolecular PathologyP53

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Area of Science:

  • Molecular Biology
  • Genetics
  • Cell Biology

Background:

  • Gene dosage significantly impacts cellular functions.
  • Studying gene dosage effects in vitro has been challenging.
  • CRISPR/Cas9 technology offers potential for creating precise gene editing models.

Purpose of the Study:

  • To refine and accelerate the development of in vitro gene dosage models.
  • To utilize CRISPR/Cas9 for sequential knockout of gene alleles.
  • To establish a rapid and efficient method for studying gene dosage.

Main Methods:

  • Employed Cas9 nuclease mRNA and fluorescently labeled tracr-guide RNA for gene editing.
  • Utilized fluorescence-activated cell sorting (FACS) for isolation of transfected cells.
  • Applied high-resolution melting (HRM) analysis for rapid screening of mutations.

Main Results:

  • Achieved a 37.5% targeting efficiency in HCT116 cells for TP53 gene editing.
  • Identified a heterozygous mutation (single base deletion) in one clone.
  • Confirmed mutation expression via mRNA sequencing and observed altered p53 protein levels.

Conclusions:

  • Successfully developed an efficient in vitro model for studying gene dosage effects.
  • The protocol is versatile and applicable to any gene.
  • Demonstrated the utility of Cas9 mRNA, labeled guide RNA, and HRM for rapid gene editing and analysis.