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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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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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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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Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9
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Protocol for HiBiT tagging endogenous proteins using CRISPR-Cas9 gene editing.

Kaylee P Lankford1, John D Hulleman2

  • 1Department of Ophthalmology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA.

STAR Protocols
|April 10, 2024
PubMed
Summary

This study introduces a new method combining CRISPR-Cas9 genome editing and the NanoBiT system for efficient in vitro and in vivo protein detection. This approach simplifies high-throughput screening of endogenous proteins.

Keywords:
Biotechnology and bioengineeringCRISPRGene ExpressionHealth SciencesHigh Throughput ScreeningMolecular BiologyProtein Biochemistry

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • Accurate protein detection is crucial for understanding cellular functions.
  • Existing methods for endogenous protein analysis can be complex and low-throughput.

Purpose of the Study:

  • To present a novel, simplified method for in vitro and in vivo protein detection.
  • To enable high-throughput screening of endogenous proteins using CRISPR-Cas9 and NanoBiT.

Main Methods:

  • Integration of CRISPR-Cas9 genome editing with the NanoBiT protein detection system.
  • Detailed protocols for cell culturing, ribonucleoprotein delivery, and cell monitoring.
  • Analysis of edits using HiBiT assays, genomic DNA analysis, and HiBiT blotting.

Main Results:

  • Successful implementation of a straightforward and multifunctional protein detection protocol.
  • Demonstration of edit specificity through various analytical techniques.
  • Facilitation of high-throughput screening capabilities for endogenous proteins.

Conclusions:

  • The presented method offers a simple, efficient, and versatile tool for protein detection.
  • This approach significantly enhances the ease of conducting high-throughput screens on endogenous proteins.