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Allosteric Proteins-ATCase01:19

Allosteric Proteins-ATCase

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Binding sites linkages can regulate a protein's function.  For example, enzyme activity is often regulated through a feedback mechanism where the end product of the biochemical process serves as an inhibitor.
Aspartate transcarbamoylase (ATCase) is a cytosolic enzyme that catalyzes the condensation of L-aspartate and carbamoyl phosphate to  N-carbamoyl-L-aspartate. This reaction is the first step in pyrimidine biosynthesis. UTP and CTP, the end products of the pyrimidine synthesis...
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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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Allosteric Regulation01:08

Allosteric Regulation

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Allosteric regulation of enzymes occurs when the binding of an effector molecule to a site that is different from the active site causes a change in the enzymatic activity. This alternate site is called an allosteric site, and an enzyme can contain more than one of these sites. Allosteric regulation can either be positive or negative, resulting in an increase or decrease in enzyme activity. Most enzymes that display allosteric regulation are metabolic enzymes involved in the degradation or...
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Ligand Binding and Linkage00:49

Ligand Binding and Linkage

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Allosteric proteins have more than one ligand binding site; the binding of a ligand to any of these sites influences the binding of ligands to the other sites. When a protein is allosteric, its binding sites are called coupled or linked.  In the case of enzymes, the site that binds to the substrate is known as the active site and the other site is known as the regulatory site. When a ligand binds to the regulatory site, this leads to conformational changes in the protein that can influence...
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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.
The recognition sites for Cre recombinase called LoxP...
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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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Secondary Conformational Checkpoint in CRISPR-Cas9.

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Identification of a Cryptic Binding Site in CRISPR-Cas9 for Targeted Inhibition.

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Related Experiment Video

Updated: Jan 13, 2026

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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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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Differential Allosteric Modulation of Cas9 Specificity.

Yuanhao Li1, Xin Li1, Yingjie Chen1

  • 1College of Chemistry and Chemical Engineering, Shanghai University of Engineering Science, Shanghai 201620, China.

Journal of Chemical Theory and Computation
|January 9, 2026
PubMed
Summary

CRISPR-Cas9 gene editing precision is improved by altering guide RNAs (gRNAs) or Cas9. These changes, though distant from the active site, allosterically control Cas9 by immobilizing the HNH nuclease domain, enhancing editing specificity.

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Area of Science:

  • Molecular Biology
  • Biophysics
  • Genomics

Background:

  • CRISPR-Cas9 gene editing relies on precise targeting, but off-target cleavage remains a challenge.
  • Strategies like modified guide RNAs (gRNAs) and Cas9 variants aim to enhance specificity.
  • The molecular mechanisms underlying how distant modifications affect Cas9 activity are not fully understood.

Purpose of the Study:

  • To elucidate the molecular mechanisms by which PAM-distal alterations modulate CRISPR-Cas9 activity and specificity.
  • To investigate how gRNA truncation, base mismatching, and Cas9 mutations impact Cas9 conformational dynamics and allosteric regulation.
  • To establish a dynamic framework linking distal structural perturbations to Cas9 activity control.

Main Methods:

  • Near-millisecond all-atom molecular dynamics simulations were employed.
  • Simulations analyzed the effects of diverse PAM-distal perturbations on Cas9.
  • Conformational dynamics and allosteric regulation of Cas9 were investigated.

Main Results:

  • All tested perturbations, including gRNA truncation and base mismatching, impede the transition to the catalytically active state by altering HNH dynamics.
  • Distinct allosteric routes were identified for different perturbations, including REC3 reorientation and L2 linker engagement.
  • Evolved mutations were found to remodel global motional modes, constraining HNH flexibility.

Conclusions:

  • Multiple structural pathways converge on HNH immobilization and catalytic suppression, unifying the effects of RNA, DNA, and protein modifications.
  • This study provides mechanistic insights into Cas9 fidelity regulation.
  • The findings offer principles for designing next-generation, high-precision genome editors.