METHODS AND COMPOSITIONS FOR DETECTING A TARGET RNA
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.
1 - 90 . (canceled)
91 . A method of detecting a single-stranded target RNA in a biological sample comprising 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 non-target RNA by the Type VI CRISPR RNA-guided RNA endonuclease.
92 . The method according to claim 91 , wherein the Type VI CRISPR RNA-guided RNA endonuclease cleaves at least 50% of the RNAs present in the biological sample within 1 hour said contacting.
93 . The method according to claim 91 , 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.
94 . The method according to claim 91 , wherein the single-stranded target RNA is present in the biological sample in a range of from 10 fM to 1 nM.
95 . The method according to claim 91 , 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.
96 . The method according to claim 95 , wherein the Type VI CRISPR RNA-guided RNA endonuclease cleaves 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 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.
97 . The method according to claim 91 , 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.
98 . The method according to claim 91 , wherein the sample is contacted for 1 hour or less prior to said measuring.
99 . The method according to claim 91 , comprising determining an amount of 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 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 target RNA present in the sample.
100 . The method according to claim 91 , wherein the sample comprises from 10 to 10 6 RNAs that differ from one another in sequence.
101 . The method according to claim 91 , wherein measuring a 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.
102 . The method according to claim 91 , wherein:
(i) the method comprises contacting the biological sample with a labeled non-target RNA comprising 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.
103 . The method of according to claim 102 , wherein the labeled non-target RNA comprises a fluorescence resonance energy transfer (FRET) pair a quencher/fluor pair.
104 . The method of according to claim 102 , 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.
105 . The method according to claim 91 , 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).
106 . The method according to claim 91 , 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.
107 . The method according to claim 91 , wherein the sample comprises RNAs from a cell lysate.
108 . The method according to claim 91 , 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.
109 . The method according to claim 91 , further comprising lysing the sample prior to the contacting.
110 . The method according to claim 108 , wherein the Type VI CRISPR RNA-guided RNA endonuclease is not denatured prior to the measuring.