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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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Genome Editing in Human Cells Using CRISPR/Cas Nucleases.

Nicolas Wyvekens1, Shengdar Q Tsai1,2, J Keith Joung1,2

  • 1Molecular Pathology Unit, Center for Computational and Integrative Biology and Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts.

Current Protocols in Molecular Biology
|October 2, 2015
PubMed
Summary

This study details protocols for CRISPR/Cas genome editing, highlighting its ease of use compared to older methods. It includes methods for designing CRISPR tools and enhancing their specificity for precise gene modification.

Keywords:
CRISPRCas9FokI-dCas9RNA-guided FokI nucleasesgenome editinghuman cellstru-gRNA

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR-associated (Cas) system is a powerful genome editing tool.
  • CRISPR/Cas technology offers advantages over older methods like zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) due to simpler requirements.
  • Its adoption is widespread across various model organisms and human cell types.

Purpose of the Study:

  • To provide detailed protocols for utilizing the CRISPR/Cas system for genome editing.
  • To guide researchers in designing and cloning vectors for single or multiplex guide RNAs (gRNAs).
  • To offer methods for transient transfection of human cell lines and mutation frequency quantification.

Main Methods:

  • Protocols for designing and cloning vectors expressing single or multiplex gRNAs.
  • Methods for transient transfection of human cell lines.
  • Quantitation of mutation frequencies using the T7 endonuclease I assay.
  • Implementation of truncated gRNAs and dimeric RNA-guided FokI nucleases to enhance specificity.

Main Results:

  • Established protocols enable efficient genome editing using the CRISPR/Cas system.
  • The described methods facilitate the design and application of CRISPR/Cas tools for various research needs.
  • Enhanced specificity was achieved using truncated gRNAs and dimeric RNA-guided FokI nucleases.

Conclusions:

  • The CRISPR/Cas system provides an accessible and efficient method for genome editing.
  • The provided protocols and specificity-enhancing techniques empower researchers to perform precise genetic modifications.
  • This work facilitates broader application of CRISPR/Cas technology in biological research.