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US 12,742,196 B2Granted
Methods and compositions for detecting a target RNA
AssigneeThe Regents of the University of California
InventorsJennifer A. Doudna, Mitchell Ray O'Connell, Alexandra East-Seletsky, Spencer Charles Knight, James Harrison Doudna Cate
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Abstract
The present disclosure provides methods for detecting a single-stranded target RNA. The present disclosure provides methods of cleaving a precursor C2c2 guide RNA array into two or more C2c2 guide RNAs. The present disclosure provides a kit for detecting a target RNA in a sample.
Claims19
A method of detecting a single-stranded target RNA present in a biological sample at a concentration of from 10 fM to 1 nM, wherein the biological sample comprises a plurality of RNAs, the method comprising:
- a) contacting the biological sample with:
- (i) a Type VI CRISPR RNA-guided RNA endonuclease having distinct ribonuclease activities for (1) CRISPR RNA processing and (2) single-stranded RNA degradation,
- (ii) a guide RNA comprising a guide sequence that hybridizes to a target sequence of the single-stranded target RNA and a region that binds to the Type VI CRISPR RNA-guided RNA endonuclease, and
- (iii) a labeled non-target RNA, wherein the Type VI CRISPR RNA-guided RNA endonuclease cleaves the labeled non-target RNA when the guide RNA hybridizes to the target sequence;
- and
- b) measuring a detectable signal indicative of cleavage of the labeled non-target RNA by the Type VI CRISPR RNA-guided RNA endonuclease,
- wherein the detectable signal is indicative of the presence of the single-stranded target RNA in the biological sample.
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Dependencies are inferred from the claim text.