Methods and Compositions for Using Argonaute to Modify a Single-Stranded Target Nucleic Acid

Patent Status

Brief Description

Argonaute (Ago) proteins are small RNA or DNA guided, site-specific endonucleases, which are present in all three kingdoms of life. The various functions of Argonaute proteins in eukaryotes have been studied extensively.  Recent studies have suggested that prokaryotic Argonautes are involved in identifying foreign genetic elements in a sequence specific manner and/or in the recruitment of nucleases.  The nucleic acid guided binding and cleavage activities of Argonaute (Ago) proteins are reminiscent of the activities of RNA-guided proteins within CRISPR-Cas systems and use a guide nucleic acid (e.g., a guide RNA) to identify a target nucleic acid. The guide RNAs utilized by all currently known Ago proteins include a 5’-phosphate and a 3’-hydroxl and these methods are limited because the guide RNAs utilized by the heterologously expressed Ago proteins are generally indistinguishable from the thousands of RNAs present in the host cell.

UC Berkeley researchers have discovered a technology that facilitates the precise and controlled targeting of Argo nuclease activity (or other protein activities such as binding) to single stranded target nucleic acids (e.g., ssRNA, ssDNA, mRNA, rRNA, tRNA, microRNA, etc.) which overcomes the above-mentioned limitation.  The researchers showed CRISPR-associated Ago proteins cleaved single-stranded target sequences using 5’-hydroxylated guide RNAs rather than the 5-phoshorylated guides used by known Argonautes.  

Suggested uses

• Precise and controlled targeting of Ago nuclease activity (or binding/cleaving) to single stranded target nucleic acids (e.g., ssRNA, ssDNA, mRNA, rRNA, tRNA, etc).

Advantages

• Guide RNAs distinguishable from the thousands of RNAs present in the host cell

Publication

A bacterial Argonaute with noncanonical guide RNA specificity