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Related Concept Videos

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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CRISPR01:59

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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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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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Related Experiment Video

Updated: Dec 9, 2025

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells
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Gene Disruption Using Chemically Modified CRISPR-Cpf1 RNA.

Moira A McMahon1, Meghdad Rahdar2

  • 1Ionis Pharmaceuticals Inc., Carlsbad, CA, USA.

Methods in Molecular Biology (Clifton, N.J.)
|September 14, 2020
PubMed
Summary

This study details a protocol for CRISPR-Cpf1 gene editing in mammalian cells using chemically modified guide RNAs. This method enhances efficiency and reduces off-target effects for precise genetic modification.

Keywords:
CRISPR-Cpf1Cas12aChemically modified nucleic acidGene editingGuide RNASurveyor nuclease assaySynthetic CRISPR RNA

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Last Updated: Dec 9, 2025

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Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms
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Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • CRISPR gene editing is a vital tool for modifying mammalian DNA.
  • Diverse CRISPR systems and chemically modified guide RNAs enhance editing capabilities.
  • CRISPR-Cpf1 offers reduced off-target cleavage compared to CRISPR-Cas9.

Purpose of the Study:

  • To provide a detailed protocol for CRISPR-Cpf1 gene editing in cell lines.
  • To optimize gene editing efficiency using chemically modified guide RNAs.
  • To facilitate precise genomic modifications for research and therapeutic applications.

Main Methods:

  • Designing chemically modified guide RNAs for CRISPR-Cpf1.
  • Introducing CRISPR-Cpf1 and guide RNAs into mammalian cell lines.
  • Analyzing gene editing events for efficiency and specificity.

Main Results:

  • Demonstrated successful implementation of CRISPR-Cpf1 gene editing.
  • Showcased improved editing efficiency with chemically modified guide RNAs.
  • Validated the protocol for site-specific gene modification in cell lines.

Conclusions:

  • The protocol enables efficient and specific gene editing using CRISPR-Cpf1.
  • Chemically modified guide RNAs are crucial for enhancing CRISPR-Cpf1 performance.
  • This method advances the application of gene editing in mammalian cells.