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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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Substrate Generation for Endonucleases of CRISPR/Cas Systems
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A Non-Stem-Loop CRISPR RNA Is Processed by Dual Binding Cas6.

Yaming Shao1, Hagen Richter2, Shengfang Sun1

  • 1Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306, USA.

Structure (London, England : 1993)
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Researchers discovered how Cas6 enzymes process CRISPR RNA. Two Cas6 proteins bind to specific RNA motifs, enabling cleavage of non-stem-loop CRISPR RNA structures.

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Biochemistry

Background:

  • CRISPR-Cas systems provide adaptive immunity in bacteria.
  • Cas6 endoribonucleases process CRISPR RNA (crRNA) for system function.
  • A specific subclass of crRNA lacks typical stem-loop structures, posing a processing challenge.

Purpose of the Study:

  • To elucidate the mechanism by which Cas6 enzymes recognize and process non-stem-loop CRISPR RNA.
  • To determine the structural basis for Cas6-crRNA interaction and cleavage.

Main Methods:

  • Cocrystallography of Methanococcus maripaludis Cas6b (MmCas6b) with its cognate repeat RNA.
  • Biochemical analyses to validate binding models and functional roles.

Main Results:

  • Cas6 employs a dual-site binding mechanism, interacting with two distinct AAYAA motifs on the repeat RNA.
  • One MmCas6b molecule binds distally, and another binds adjacent to the cleavage site.
  • The enzyme stabilizes the RNA structure at the cleavage site by mimicking a base pair, facilitating phosphodiester bond breakage.
  • This binding mode potentially outcompetes a non-productive RNA conformation.

Conclusions:

  • Cas6 processes non-stem-loop CRISPR RNA through a novel dual-site binding mechanism.
  • The protein-RNA interaction involves specific AAYAA motif recognition and a crucial base-pair mimicry at the cleavage site.
  • This study reveals a new pathway for CRISPR RNA maturation in bacteria.