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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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Mismatch type impacts interference and priming activities in the type I-E CRISPR-Cas system.

Phong T Phan1, Meric Ozturk1, Elizabeth M Dougherty1

  • 1Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, Iowa, USA.

The Journal of Biological Chemistry
|March 27, 2026
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Summary

The crRNA sequence significantly impacts the Type I-E CRISPR-Cas system's ability to combat foreign DNA, even when target DNA mutations occur. Understanding this interplay is key for CRISPR immunity research.

Keywords:
CRISPR–CasDNADNA enzymeDNA–protein interactioncrRNA

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Area of Science:

  • Molecular Biology
  • Genetics
  • Immunology

Background:

  • Type I-E CRISPR-Cas systems use Cascade and Cas3 to target foreign nucleic acids.
  • Priming allows rapid adaptation by acquiring new spacers after DNA targeting.
  • Mutations in the PAM or seed region can affect interference and priming.

Purpose of the Study:

  • To systematically investigate the influence of crRNA seed sequences on mutational tolerance in Type I-E CRISPR-Cas systems.
  • To clarify the role of crRNA sequence variations in CRISPR immunity outcomes.

Main Methods:

  • Engineered four E. coli strains with distinct crRNA spacer sequences.
  • Tested CRISPR interference and priming against a plasmid library for each strain.
  • Utilized in vitro biochemistry to analyze Cascade conformation and Cas3 recruitment.

Main Results:

  • Mutations to C or G in the seed region were often deleterious, particularly at positions 1, 2, and 4.
  • The specific crRNA sequence significantly modulated the severity of defects caused by target mutations (e.g., rC-dC, rA/G-dG).
  • Mismatch type at the seed's first position altered Cascade conformation, reducing target binding and Cas3 recruitment.

Conclusions:

  • Nucleotide identity in target mutations is crucial for Type I-E CRISPR immunity.
  • The crRNA sequence plays a critical role in determining immune outcomes when target DNA is mutated.
  • This highlights a complex interplay between target and guide sequences in CRISPR-based defense.