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

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.