Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence

Area of Science:

• Molecular Biology
• Microbial Immunity
• Genetics

Background:

• Prokaryotic CRISPR/Cas systems confer adaptive immunity against viruses.
• Viral escape from CRISPR/Cas resistance is often mediated by protospacer mutations.

Purpose of the Study:

• To investigate the specificity requirements of the Escherichia coli CRISPR/Cas system for protospacer recognition.
• To understand how mutations in viral protospacers affect CRISPR/Cas-mediated immunity.

Main Methods:

• Analysis of crRNA-protospacer binding affinity.
• Assessing the impact of protospacer mutations on CRISPR/Cas immunity in E. coli.

Main Results:

• A strict seven-nucleotide seed region in the protospacer, adjacent to the protospacer-adjacent motif, is critical for crRNA matching.
• Mutations within this seed region abolish CRISPR/Cas immunity by reducing Cascade complex binding.
• Mutations outside the seed region do not lead to viral escape, indicating relaxed specificity.

Conclusions:

• The crRNA seed sequence is crucial for efficient initial scanning of viral DNA by the E. coli Cascade complex.
• Relaxed specificity in protospacer recognition limits viral escape and broadens the targeting range of CRISPR/Cas systems.