RNA-dependent RNA targeting by CRISPR-Cas9.

Area of Science:

• Molecular Biology
• Microbiology
• Genetics

Background:

• Type II CRISPR-Cas systems provide bacterial adaptive immunity through Cas9 binding and cleavage of double-stranded DNA (dsDNA).
• Cas9 is traditionally understood to recognize DNA exclusively as its natural substrate, defending against phages and plasmids.

Purpose of the Study:

• To investigate whether Cas9 enzymes can recognize and cleave single-stranded RNA (ssRNA).
• To explore the potential of RNA-guided RNA cleavage by Cas9 for novel applications in gene regulation and defense.

Main Methods:

• Utilized Cas9 enzymes from subtypes II-A and II-C.
• Demonstrated RNA-guided RNA cleavage independent of protospacer-adjacent motif (PAM) sequences.
• Evaluated Cas9-mediated gene expression repression in bacteria.

Main Results:

• Cas9 enzymes were shown to recognize and cleave ssRNA via an RNA-guided mechanism.
• This RNA-guided RNA cleavage was programmable, site-specific, and PAM-independent.
• Cas9 activity was successfully used to reduce ssRNA phage infection in vivo.
• Cas9 mediated PAM-independent repression of bacterial gene expression.

Conclusions:

• A subset of Cas9 enzymes possess dual activity, capable of targeting both DNA and RNA.
• This finding opens avenues for programmable RNA targeting applications, expanding the utility of CRISPR-Cas technology.