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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • CRISPR RNA-guided RNase (Cas13) targets RNA, but its mechanism with structured RNAs is unclear.
  • Cas13 is activated by CRISPR RNA binding to a complementary target RNA protospacer.
  • Cellular RNA environments are highly structured, posing challenges for Cas13 activity.

Purpose of the Study:

  • To systematically investigate the impact of RNA secondary structure on Cas13 activity.
  • To elucidate the mechanism by which Cas13 interacts with structured RNAs.
  • To develop structure-informed Cas13 applications for enhanced diagnostics.

Main Methods:

  • Systematic probing of Cas13 activity against various RNA secondary structures.
  • Development of a strand displacement framework to explain protospacer inhibition.
  • Engineering an 'occluded' Cas13 variant to improve mismatch discrimination.

Main Results:

  • Secondary structure within the protospacer and downstream inhibits Cas13.
  • A strand displacement mechanism quantitatively explains protospacer structure effects.
  • Occluded Cas13 enhances mismatch discrimination up to 50-fold.
  • Occluded Cas13 enables sequence-agnostic mutation identification at low allele frequencies.

Conclusions:

  • Mechanistic understanding of Cas13 reveals structure-dependent activity.
  • Strand displacement is key to understanding Cas13 inhibition by structured RNA.
  • Occluded Cas13 expands RNA diagnostics and enables structure-informed Cas13 approaches.
  • Identified clinically relevant mutations in SARS-CoV-2, influenza viruses, and KRAS using occluded Cas13.