Granted Patent B2

US 12,742,196 · App. 18/492,550 · Granted Sep 22, 2026

Inventors: Jennifer A. Doudna (Berkeley, CA); Mitchell Ray O'Connell (Oakland, CA); Alexandra East-Seletsky (San Francisco, CA); Spencer Charles Knight (Berkeley, CA); James Harrison Doudna Cate (Berkeley, CA)

Assignee: The Regents of the University of California

C12Q1/6823C12N9/222Y02A50/30

Abstract

Claims (39)

1 . 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.

2 . The method according to claim 1 , wherein the Type VI CRISPR RNA-guided RNA endonuclease cleaves at least 50% of the RNAs present in the biological sample within 1 hour of said contacting.

3 . The method according to claim 1 , wherein the Type VI CRISPR RNA-guided RNA endonuclease cleaves from 50% to more than 90% of the RNAs present in the biological sample within 1 minute of said contacting.

4 . The method according to claim 1 , wherein the Type VI CRISPR RNA-guided RNA endonuclease is not a Leptotrichia shahii (Lsh) C2c2 protein comprising an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:3.

5 . The method according to claim 4 , wherein the Type VI CRISPR RNA-guided RNA endonuclease cleaves the labeled non-target RNA at least 1.2-fold more efficiently than a Leptotrichia shahii (Lsh) C2c2 protein comprising at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:3 or wherein the Type VI CRISPR RNA-guided RNA endonuclease cleaves the labeled non-target RNA at least 1.5-fold more efficiently than a Leptotrichia shahii (Lsh) C2c2 protein comprising the amino acid sequence set forth in SEQ ID NO:3.

6 . The method according to claim 1 , wherein the Type VI CRISPR RNA-guided RNA endonuclease comprises:

(i) an amino acid sequence having 80% or more amino acid sequence identity with the amino acid sequence set forth in any one of SEQ ID NOs: 1, 2, 4-6, 301, 304, 307, 310, 312-314, 316, or 321;

(ii) the amino acid sequence set forth in any one of SEQ ID NOs: 1, 2, 4-6, 301, 304, 307, 310, 312-314, 316, or 321;

(iii) an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:2, and comprises a substitution of one or more of R472, H477, R1048, and H1053;

(iv) an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:2, and comprises a substitution of amino acids R472 and H477; or

(v) an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:2, and comprises a substitution of amino acids R1048 and H1053.

7 . The method according to claim 1 , wherein the sample is contacted for 1 hour or less prior to said measuring.

8 . The method according to claim 1 , comprising determining an amount of the single-stranded target RNA present in the sample and, optionally, wherein said determining comprises:

(a) measuring the detectable signal to generate a test measurement; measuring a detectable signal produced by a reference sample to generate a reference measurement; and comparing the test measurement to the reference measurement to determine an amount of the single-stranded target RNA present in the sample, or

(b) measuring the detectable signal to generate a test measurement; measuring a detectable signal produced by each of two or more reference samples, wherein the two or more reference samples each include a different amount of a positive control RNA, to generate two or more reference measurements; and comparing the test measurement to the two or more reference measurements to determine an amount of the single-stranded target RNA present in the sample.

9 . The method according to claim 1 , wherein the plurality of RNAs comprises from 10 to 10 6 RNAs that differ from one another in sequence.

10 . The method according to claim 1 , wherein measuring the detectable signal comprises one or more of: visual based detection, sensor based detection, color detection, gold nanoparticle based detection, fluorescence polarization, colloid phase transition/dispersion, electrochemical detection, fluorescent signal detection, and semiconductor-based sensing.

11 . The method according to claim 1 , wherein:

(i) the labeled non-target RNA comprises a fluorescence-emitting dye pair, and

(ii) the detectable signal is produced by the fluorescence-emitting dye pair upon cleavage of the labeled non-target RNA by the Type VI CRISPR RNA-guided RNA endonuclease.

12 . The method of according to claim 11 , wherein the labeled non-target RNA comprises a fluorescence resonance energy transfer (FRET) pair or a quencher/fluor pair.

13 . The method of according to claim 11 , wherein:

(i) the labeled non-target RNA produces an amount of detectable signal prior to being cleaved, and the amount of detectable signal is reduced when the labeled non-target RNA is cleaved,

(ii) the labeled non-target RNA produces a first detectable signal prior to being cleaved and a second detectable signal when the labeled non-target RNA is cleaved,

(iii) the detectable signal is produced when the labeled non-target RNA is cleaved, or

(iv) an amount of detectable signal increases when the labeled non-target RNA is cleaved.

14 . The method according to claim 1 , wherein the labeled non-target RNA comprises one or more of: a non-natural internucleoside linkage, a nucleic acid mimetic, a modified nucleobase, a

modified sugar moiety, a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a morpholino nucleic acid, and a cyclohexenyl nucleic acid (CeNA).

15 . The method according to claim 1 , wherein said contacting is carried out in an acellular sample or wherein said contacting is carried out in a cell in vitro, ex vivo, or in vivo.

16 . The method according to claim 1 , wherein the sample comprises RNAs from a cell lysate.

17 . The method according to claim 1 , wherein the measuring comprises measuring a change in the detectable signal over a period of time or wherein the measuring comprises measuring a change in the detectable signal in real time.

18 . The method according to claim 1 , further comprising lysing the sample prior to the contacting.

19 . The method according to claim 17 , wherein the Type VI CRISPR RNA-guided RNA endonuclease is not denatured prior to the measuring.

Assignments (1)

Continuity (6)

References Cited (264)