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Related Concept Videos

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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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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Homologous Recombination02:31

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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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Updated: May 22, 2025

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Insights into the compact CRISPR-Cas9d system.

Jie Yang1,2, Tongyao Wang1,2, Ying Huang3,4,5

  • 1State Key Laboratory of Experimental Hematology, Tianjin Institute of Immunology, The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China.

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The smallest Cas9d protein uses a compact structure for DNA cutting. Its guide RNA and protein work together to precisely recognize targets, enabling development of high-fidelity mini-CRISPR tools.

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Area of Science:

  • Molecular Biology
  • Structural Biology
  • Biochemistry

Background:

  • Cas9d is the smallest known Cas9 enzyme, but its DNA cleavage mechanism is not well understood.
  • Understanding Cas9d's mechanism is crucial for developing novel CRISPR tools.

Purpose of the Study:

  • To elucidate the structural and mechanistic basis of target recognition and DNA cleavage by Cas9d.
  • To investigate the role of sgRNA in Cas9d function and regulation.
  • To engineer a more compact and efficient Cas9d system.

Main Methods:

  • Cryo-electron microscopy (cryo-EM) to determine structures of Cas9d-sgRNA complexes.
  • Biochemical assays to study PAM recognition and DNA cleavage.
  • Structural comparisons with other Cas9 variants.
  • Structure-guided protein and sgRNA engineering.

Main Results:

  • Determined cryo-EM structures of Cas9d-sgRNA in target-free and target-bound states.
  • Identified a 17-base pair heteroduplex requirement for nuclease activity.
  • Revealed a hybrid functional module involving sgRNA and REC domain for target recognition.
  • Demonstrated lower mismatch tolerance in Cas9d compared to SpyCas9.
  • Engineered a more compact Cas9d system with retained nuclease activity.

Conclusions:

  • Cas9d utilizes a unique mechanism involving sgRNA-REC domain interaction for precise target recognition.
  • Cas9d exhibits high fidelity due to stringent heteroduplex monitoring.
  • Structure-guided engineering can yield improved mini-CRISPR systems.
  • Findings provide a foundation for developing advanced, high-fidelity gene editing tools.