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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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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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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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Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins
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Creating a Modular Activatable CRISPR-Cas12a System by Engineering crRNA Scaffold with a Steric Hindrance Effector.

Pengfei Liu1, Sheng Li1, Jiayu Zeng1

  • 1Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research, Ministry of Education, Institute of Interdisciplinary Studies, College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, China.

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This study introduces a new CRISPR-Cas12a system controlled by steric hindrance. The system can be activated by light or enzymes, enabling precise gene editing and diagnostics.

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Gene Editing Technologies

Background:

  • CRISPR-Cas systems lack precise spatiotemporal control, limiting their accuracy and efficiency in applications.
  • Developing modular, stimulus-responsive CRISPR platforms remains a significant challenge.

Purpose of the Study:

  • To engineer a modular, activatable CRISPR-Cas12a system with spatiotemporal control.
  • To demonstrate the system's responsiveness to diverse stimuli, including light, enzymes, and chemicals.
  • To enable advanced biotechnological applications like nucleic acid diagnostics and targeted imaging.

Main Methods:

  • Engineered a CRISPR-Cas12a system using a crRNA scaffold with a stimulus-cleavable linker and a steric hindrance effector (SHE) motif.
  • Systematically evaluated various SHE designs (linear, duplex, hairpin, triplex) for inhibitory effects.
  • Validated system activation via ultraviolet light, enzymatic cleavage (APE1), and chemical triggers (GSH).

Main Results:

  • Identified SHEs that effectively inhibit CRISPR-Cas12a activity in a steric hindrance-dependent manner.
  • Demonstrated successful release of SHEs upon UV light exposure, restoring Cas12a functionality.
  • Showcased the system's adaptability to other Cas12a orthologs and its response to enzymatic and chemical stimuli.
  • Achieved light-activatable nucleic acid diagnostics and APE1-activatable, tumor cell-specific microRNA imaging.

Conclusions:

  • Developed an innovative modular activatable CRISPR-Cas12a system using steric hindrance for precise control.
  • The system offers modularity and adaptability to various stimuli, overcoming limitations of traditional CRISPR systems.
  • This platform provides a foundation for designing next-generation, controllable CRISPR-based tools for diagnostics and therapeutics.