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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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CRISPR and crRNAs02:53

CRISPR and crRNAs

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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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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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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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Computational Methods to Engineer Cas Proteins for Efficient Genome Editing.

Muhammad Qaiser Fatmi1, Aneeqa Nadeem2, Mehraj Abbasov3

  • 1Department of Biosciences, COMSATS University Islamabad, Islamabad, Pakistan. qaiser.fatmi@comsats.edu.pk.

Methods in Molecular Biology (Clifton, N.J.)
|November 1, 2025
PubMed
Summary
This summary is machine-generated.

Researchers can now rationally design improved CRISPR/Cas genome editing tools. This protocol enhances Cas protein stability and specificity using computational methods for more precise DNA targeting.

Keywords:
CRISPR/Cas9Coevolutionary couplingGenome editingMolecular dynamics simulationMutant stability predictionNetwork centralityProtein engineering

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Bioinformatics

Background:

  • The CRISPR/Cas system enables programmable, sequence-specific DNA targeting for genome editing.
  • Widespread application is hindered by off-target effects and variable efficiency.
  • Need for rational design strategies to engineer enhanced CRISPR/Cas variants.

Purpose of the Study:

  • To present an integrated computational protocol for designing improved CRISPR/Cas variants.
  • To enhance Cas protein stability and specificity for genome editing.
  • To provide researchers with easy-to-follow steps for rational protein engineering.

Main Methods:

  • Coevolutionary coupling analysis to identify critical residues.
  • Mutant stability prediction for energetically favorable substitutions.
  • Network centrality analysis to assess impact on intramolecular communication.
  • Iterative application of network analysis and molecular dynamics (MD) simulations.
  • Validation of structural integrity and dynamic behavior using MD simulations.

Main Results:

  • Identification of conserved and covarying residues crucial for Cas protein function.
  • Prediction of mutations that enhance protein stability while preserving allosteric interactions.
  • Evaluation of the impact of mutations on intramolecular communication pathways.
  • Validation of engineered variants' structural integrity and dynamic behavior through MD simulations.

Conclusions:

  • The integrated multiscale computational strategy streamlines the engineering of optimized Cas proteins.
  • This approach facilitates the rational design of CRISPR/Cas variants with improved stability and specificity.
  • The protocol empowers researchers to develop more precise and efficient genome editing tools.