Methods and Compositions for Modifying a single stranded Target Nucleic Acid

Patent Status

Brief Description

The CRISPR-Cas9 RNA-guided DNA endonuclease uses RNA–DNA complementarity to identify target sites for sequence-specific double-stranded DNA cleavage. Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, known as the protospacer adjacent motif (PAM), next to and on the strand opposite the target site in dsDNA.  Cas9 has proven to be a versatile tool for genome engineering and gene regulation, but it has been thought to be incapable of targeting RNA.

UC Berkeley researchers have developed a technology and methods that allows for precise binding and/or cleaving a single stranded target nucleic acid that does not depend on the presence of PAM in the target nucleic acid.  The researches showed that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide.  Using specially designed PAM-presenting oligonucleotides (PAMmers), Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences.

Suggested uses

• Binding and/or cleaving of a single stranded nucleic acid targets, including single stranded RNA targets (e.g., ssRNA, mRNA, tRNA, microRNA, etc.)

Advantages

• Cas9 can be directed to bind or cut RNA targets while avoiding corresponding DNA sequences
• Cas9 can be used for programmable transcript recognition without the need for tags

Publication

 Programmable RNA recognition and cleavage by CRISPR/Cas9