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Related Concept Videos

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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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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: Jan 6, 2026

Gene Knock-in by CRISPR/Cas9 and Cell Sorting in Macrophage and T Cell Lines
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Optimised insert design for improved single-molecule imaging and quantification through CRISPR-Cas9 mediated

Abdullah O Khan1, Carl W White2,3,4,5, Jeremy A Pike6,2

  • 1Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, B15 2TT, UK. A.Khan.4@bham.ac.uk.

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Summary

CRISPR-Cas9 genome editing enables precise endogenous protein labeling for advanced microscopy. Optimizing fluorescent protein inserts improves expression and enables accurate single-molecule quantification, overcoming classical limitations.

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Microscopy

Background:

  • Classical limitations in single molecule localization microscopy hinder accurate protein analysis.
  • CRISPR-Cas9 genome editing offers a novel approach for endogenous protein tagging.

Purpose of the Study:

  • To systematically compare different CRISPR-Cas9 knock-in strategies for optimizing endogenous protein labeling.
  • To evaluate the impact of insert variants on protein expression and function for single molecule imaging.

Main Methods:

  • CRISPR-Cas9 genome editing for knock-in of fluorescent protein variants.
  • Systematic comparison of mEos variants at the TubA1B locus.
  • Application to G protein-coupled receptor (GPCR) CXCR4 labeling.
  • Analysis of protein expression, localization, and function.

Main Results:

  • Monomeric and codon-optimized mEos variants enhanced expression at the TubA1B locus.
  • Insert design significantly affected CXCR4 expression, localization, and function.
  • Endogenously labeled CXCR4 allowed accurate single-molecule quantification upon ligand treatment, unlike over-expressed samples.

Conclusions:

  • Optimized CRISPR-Cas9 knock-in strategies significantly improve endogenous protein labeling for microscopy.
  • Endogenous labeling with CRISPR-Cas9 provides substantial quantitative benefits for studying protein dynamics.
  • CRISPR-mediated protein labeling is a powerful tool for advancing single-molecule imaging and quantitative biology.