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Related Experiment Video

Updated: Feb 7, 2026

Substrate Generation for Endonucleases of CRISPR/Cas Systems
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Mismatch type impacts interference and priming activities in the type I-E CRISPR-Cas system.

Phong T Phan1,2,3, Meric Ozturk1,2, Elizabeth M Dougherty1

  • 1Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, USA.

Biorxiv : the Preprint Server for Biology
|February 6, 2026
PubMed
Summary

The CRISPR-Cas system

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Genetics

Background:

  • Type I-E CRISPR-Cas systems use Cascade and Cas3 for RNA-guided DNA targeting.
  • Priming allows for rapid acquisition of new spacers within the CRISPR array.
  • Mutations in the PAM and seed regions can affect interference and priming.

Purpose of the Study:

  • To investigate the impact of crRNA spacer sequence on mutational tolerance in Type I-E CRISPR-Cas systems.
  • To systematically test the effects of crRNA seed sequences on CRISPR interference and priming.

Main Methods:

  • Engineered four E. coli strains with variable spacer sequences.
  • Tested CRISPR interference and priming against a plasmid library for each strain.
  • Performed in vitro biochemistry to analyze Cas8 conformation and binding rates.

Main Results:

  • Mutations to C or G in the seed region were deleterious, especially at positions 1, 2, and 4.
  • The crRNA sequence significantly impacted the level of defect, with rC-dC and rA/G-dG causing the largest defects.
  • Mismatch type at the seed's first position affected Cas8 conformation, reducing Cascade-target binding and Cas3 recruitment.

Conclusions:

  • Nucleotide identity of target mutations is a key factor in Type I-E CRISPR immunity.
  • The crRNA sequence also plays a crucial role in determining immune outcomes upon target mutation.
  • Understanding these interactions is vital for optimizing CRISPR-based technologies.