Noncanonical crRNAs derived from host transcripts enable multiplexable RNA detection by Cas9.

Area of Science:

• Molecular Biology
• CRISPR-Cas Systems
• Diagnostic Technology

Background:

• CRISPR-Cas systems use CRISPR RNAs (crRNAs) to identify foreign genetic material.
• Type II CRISPR-Cas systems rely on trans-activating crRNA (tracrRNA) for crRNA processing and Cas9 enzyme utilization.
• tracrRNA typically hybridizes with specific crRNAs.

Purpose of the Study:

• To investigate the interaction of Cas9-RNA complexes from Campylobacter jejuni.
• To discover novel crRNA formation mechanisms and their potential for DNA targeting.
• To engineer a versatile RNA detection platform based on modified tracrRNAs.

Main Methods:

• Analysis of Cas9-RNA complexes from Campylobacter jejuni.
• Discovery of tracrRNA hybridizing to endogenous cellular RNAs, forming noncanonical crRNAs.
• Engineering of reprogrammed tracrRNAs to link RNA presence with Cas9-mediated DNA targeting.
• Development of the LEOPARD (leveraging engineered tracrRNAs and on-target DNAs for parallel RNA detection) platform.

Main Results:

• Identified noncanonical crRNAs generated by tracrRNA hybridization to cellular RNAs, capable of guiding Cas9 DNA targeting.
• Engineered tracrRNAs enabled DNA targeting based on the presence of any desired RNA.
• The LEOPARD platform achieved simultaneous detection of multiple viral RNAs in a single test.
• LEOPARD distinguished between SARS-CoV-2 and its D614G variant with single-nucleotide precision in patient samples.

Conclusions:

• The discovery of noncanonical crRNA formation expands the understanding of CRISPR-Cas system versatility.
• Engineered tracrRNAs provide a novel mechanism for RNA-guided DNA targeting.
• The LEOPARD platform offers a powerful, multiplexable tool for sensitive and specific RNA detection, including viral diagnostics.