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Programmable RNA recognition and cleavage by CRISPR/Cas9.

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This summary is machine-generated.

CRISPR-Cas9 can now target RNA, not just DNA. By using a separate DNA sequence (PAMmer), Cas9 can bind and cut specific RNA molecules, enabling programmable transcript recognition.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • CRISPR-associated protein 9 (Cas9) is a DNA endonuclease guiding target recognition via RNA complementarity and a protospacer adjacent motif (PAM).
  • Cas9 is widely used for genome engineering but was considered unable to target RNA.
  • Understanding Cas9's substrate specificity is crucial for expanding its applications.

Purpose of the Study:

  • To investigate if Cas9 can target single-stranded RNA (ssRNA) with the assistance of a PAM sequence.
  • To explore the mechanism of Cas9-RNA interaction and cleavage.
  • To demonstrate the utility of Cas9 for RNA targeting in a biological context.

Main Methods:

  • In vitro assays using Cas9, guide RNA, ssRNA targets, and trans-presented PAM oligonucleotides (PAMmers).
  • Characterization of Cas9 binding affinity to ssRNA targets with PAMmers.
  • Demonstration of site-specific ssRNA cleavage stimulated by PAMmers.
  • Application of the method for isolating endogenous messenger RNA (mRNA) from cells.

Main Results:

  • Cas9 binds with high affinity to ssRNA targets when a PAM sequence is provided in trans via a DNA oligonucleotide (PAMmer).
  • PAMmers stimulate Cas9-mediated, site-specific endonucleolytic cleavage of ssRNA targets.
  • Cas9 can be directed to RNA targets while avoiding DNA sequences using specifically designed PAMmers.
  • This strategy successfully enabled the isolation of a specific endogenous mRNA from cells.

Conclusions:

  • Cas9's PAM binding is fundamentally linked to its substrate selection, enabling RNA targeting.
  • Cas9 can be programmed for specific transcript recognition and cleavage without requiring target tags.
  • This work expands the utility of Cas9 beyond DNA, opening new avenues for RNA manipulation and analysis.