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Related Concept Videos

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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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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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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Transcriptional regulators bind to specific cis-regulatory sequences in the DNA to regulate gene transcription. These cis-regulatory sequences are very short, usually less than ten nucleotide pairs in length. The short length means that there is a high probability of the exact same sequence randomly occurring throughout the genome.  Since regulators can also bind to groups of similar sequences, this further increases the chances of random binding. Transcriptional regulators form...
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Gene transcription is regulated by the synergistic action of several proteins that form a complex at a gene regulatory site. This is observed in eukaryotes, where the regulation of gene expression is a complex process. Regulatory proteins in eukaryotes can broadly be classified into two types – regulators that bind directly to specific DNA sequences and co-regulators that associate with regulatory proteins but cannot directly bind to the DNA. These co-regulators are further divided into...
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Transcription activators are proteins that promote the transcription of genes from DNA to RNA. In most cases, these proteins contain two separate domains ‒ a domain that binds to DNA and a domain for activating transcription; however, in some cases, a single domain is responsible for both binding and activation of transcription, as seen in the glucocorticoid receptor and MyoD.
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Related Experiment Video

Updated: Jun 15, 2025

Dissection of Enhancer Function Using Multiplex CRISPR-based Enhancer Interference in Cell Lines
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Specific multivalent molecules boost CRISPR-mediated transcriptional activation.

Rui Chen1,2,3,4, Xinyao Shi1,3, Xiangrui Yao1,3

  • 1Shenzhen Key Laboratory of Gene Regulation and Systems Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen, China.

Nature Communications
|August 22, 2024
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Intrinsically disordered regions (IDRs) and modular domains (MDs) can enhance CRISPR/Cas gene activation. Optimal cooperativity, not maximal, and targeting promoter-enhancer interactions are key for robust activation using these multivalent molecules.

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Area of Science:

  • Molecular Biology
  • Gene Regulation
  • Biotechnology

Background:

  • CRISPR/Cas systems offer precise gene editing capabilities.
  • Transcriptional activators fused to CRISPR/Cas components can modulate gene expression.
  • The role of intrinsically disordered regions (IDRs) and modular domains (MDs) in enhancing CRISPR/Cas activity is not fully understood.

Purpose of the Study:

  • To investigate the mechanisms by which IDRs enhance CRISPR/Cas-based transcriptional activation.
  • To explore the synergistic effects of combining IDRs and MDs with CRISPR/Cas activators.
  • To identify optimal strategies for robust gene activation using engineered CRISPR/Cas systems.

Main Methods:

  • Fusing 12 different IDRs to the dCas9-VP64 transcriptional activator.
  • Assessing the impact of IDRs on gene activation, independent of phase separation.
  • Combining dCas9-VP64-IDR constructs with modular domains (MDs).
  • Systematically varying gRNA binding sites and evaluating cis-trans cooperativity.
  • Targeting promoter-enhancer pairs and assessing chromatin interactions.

Main Results:

  • Seven out of 12 tested IDRs enhanced dCas9-VP64 activation, irrespective of phase separation.
  • MDs alone did not enhance activation but significantly boosted it when combined with dCas9-VP64-IDR.
  • Optimal cis-trans cooperativity, rather than maximal, led to the most robust gene activation.
  • Targeting promoter-enhancer regions and enhancing chromatin interactions amplified synergistic effects.

Conclusions:

  • Engineered CRISPR/Cas activators can be effectively enhanced by combining IDRs and MDs.
  • The study provides insights into the mechanisms of multivalent molecule-mediated gene activation.
  • A versatile platform for efficient gene activation using CRISPR/Cas technology has been developed.