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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 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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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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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: Jul 10, 2025

CRISPR/Cas9 Editing of the C. elegans rbm-3.2 Gene using the dpy-10 Co-CRISPR Screening Marker and Assembled Ribonucleoprotein Complexes.
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CRISPR/Cas9 Editing of the C. elegans rbm-3.2 Gene using the dpy-10 Co-CRISPR Screening Marker and Assembled Ribonucleoprotein Complexes.

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CRISPR/Cas for PET Reporter Gene Engineering.

Taemoon Chung1, Joseph R Merrill1, Scott K Lyons2

  • 1Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA.

Methods in Molecular Biology (Clifton, N.J.)
|November 25, 2023
PubMed
Summary

This study introduces a CRISPR/Cas-based method for precisely inserting PET imaging reporter transgenes into eukaryotic genomes. This technique generates more accurate reporter gene expression, enabling a new generation of gold-standard reporter transgenes.

Keywords:
CRISPRCas9DNA repairDouble-strand breakImagingKnock-inNon-homologous end joiningPETReporter transgene

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Genome Editing in Mammalian Cell Lines using CRISPR-Cas
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Genome Editing in Mammalian Cell Lines using CRISPR-Cas

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • CRISPR/Cas technology has revolutionized genome manipulation in eukaryotic cells.
  • Existing methods for reporter transgene insertion may not accurately reflect endogenous gene expression.
  • Precise genomic integration is crucial for reliable reporter gene studies.

Purpose of the Study:

  • To describe a protocol for precisely knock-in a PET imaging reporter transgene into a specific genetic locus using CRISPR/Cas.
  • To enable the generation of a new generation of "gold-standard" reporter transgenes with physiologically accurate expression.
  • To provide a detailed, three-stage experimental workflow for targeted transgene insertion.

Main Methods:

  • Utilizing CRISPR/Cas technology for precise genome editing.
  • Identifying the specific genomic locus for reporter transgene insertion.
  • Implementing a protocol for the practical insertion of the reporter transgene.
  • Developing screening methods to identify correctly targeted clones.

Main Results:

  • Demonstrated a protocol for targeted knock-in of a PET imaging reporter transgene.
  • Achieved precise integration of the reporter transgene into a specific genetic locus.
  • Established a method for screening and validating successful targeted events.
  • Showcased the potential for generating reporter transgenes with accurate physiologic expression.

Conclusions:

  • The described CRISPR/Cas protocol offers an efficient method for generating highly accurate reporter transgenes.
  • This approach facilitates the creation of "gold-standard" reporter transgenes that mimic endogenous gene expression.
  • The protocol provides a robust framework for precise genomic engineering applications in eukaryotic cells.