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Related Concept Videos

CRISPR/Cas9 Genome Editing01:28

CRISPR/Cas9 Genome Editing

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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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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

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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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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.
The CRISPR-Cas system stores a copy of foreign DNA in the host genome and uses it to identify the foreign DNA upon reinfection. CRISPR-Cas has three different...
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Related Experiment Video

Updated: Apr 20, 2026

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms
09:51

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Published on: May 25, 2018

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Protein engineering of Cas9 for enhanced function.

Benjamin L Oakes1, Dana C Nadler2, David F Savage3

  • 1Department of Molecular & Cell Biology, University of California, Berkeley, California, USA.

Methods in Enzymology
|November 16, 2014
PubMed
Summary
This summary is machine-generated.

Researchers developed a new platform to engineer novel CRISPR-Cas9 proteins for genome editing. This system enables the creation of functional Cas9 variants with enhanced capabilities for future genetic engineering tools.

Keywords:
BHCRISPRCas9DNAEMFACSGFPGene activation/repressionGenome editingHRIPTGIT dCas9LBNHEJNUCNucleasePAMPDZPIProtein engineeringRECRFPRNASOBSOCSpCas9Synthetic biologyWT dCas9aTCcrRNAdCas9sgRNAssDNAtracrRNA

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Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution
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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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Genome Editing in Mammalian Cell Lines using CRISPR-Cas

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • CRISPR/Cas systems provide defense against nucleic acid invasion in prokaryotes.
  • The Cas9 protein, derived from bacterial CRISPR/Cas systems, is a versatile tool for genome editing.
  • Current Cas9 technology faces challenges in specificity, efficiency, and controlled application.

Purpose of the Study:

  • To develop a versatile platform for engineering novel Cas9-based proteins.
  • To address limitations in current Cas9 genome editing technology.
  • To create next-generation genome-modifying tools with enhanced functionalities.

Main Methods:

  • Development of a protein engineering platform for Cas9 modification.
  • Implementation of screening and selection techniques to identify active Cas9 mutants.
  • Insertion of a heterologous PDZ domain into Cas9 to create functional variants.

Main Results:

  • Demonstration of methods for screening and selecting active Cas9 mutants.
  • Isolation of functional Cas9 variants with an inserted PDZ domain.
  • Establishment of a proof-of-concept for constructing diverse engineered Cas9 proteins.

Conclusions:

  • The developed platform facilitates the engineering of novel Cas9 functionalities.
  • This work lays the foundation for creating advanced genome editing tools.
  • Engineered Cas9 proteins hold promise for future biological research and applications.