COMPOSITIONS FOR DETECTION OF DNA AND METHODS OF USE THEREOF
Described herein are methods and systems for direct detection of DNA nucleic acids using a DNA-activated programmable RNA nuclease.
1 . A composition comprising:
a) a Type VI Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas nuclease; and
b) an engineered guide nucleic acid comprising a first segment that is reverse complementary to a segment of a target single-stranded deoxyribonucleic acid, wherein the engineered guide nucleic acid comprises a second segment that binds to the Type VI CRISPR-Cas nuclease nuclease to form a complex.
2 . The composition of claim 1 , further comprising an RNA reporter or a DNA reporter.
3 . (canceled)
4 . (canceled)
5 . The composition of claim 1 , wherein the composition further comprises the target single-stranded deoxyribonucleic acid.
6 . The composition of claim 5 , wherein the target single-stranded deoxyribonucleic acid is an amplicon of a nucleic acid.
7 . The composition of claim 6 , wherein the nucleic acid is a deoxyribonucleic acid.
8 . The composition of claim 6 , wherein the nucleic acid is a ribonucleic acid.
9 . (canceled)
10 . (canceled)
11 . The composition of claim 1 , wherein the Type VI CRISPR-Cas nuclease is a Cas13 protein.
12 . The composition of claim 11 , wherein the Cas13 protein comprises a Cas13a polypeptide, a Cas13b polypeptide, a Cas13c polypeptide, a Cas13c polypeptide, a Cas13d polypeptide, or a Cas13e polypeptide.
13 . (canceled)
14 . The composition of claim 12 , wherein the Cas13 protein comprises the Cas13a polypeptide, and wherein the Cas13a polypeptide is LbuCas13a or LwaCas13a.
15 . The composition of claim 1 , wherein the Type VI CRISPR-Cas nuclease comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NO: 18-SEQ ID NO: 35.
16 . The composition of claim 15 , wherein the Type VI CRISPR-Cas nuclease comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 19.
17 . The composition of claim 1 , wherein the composition has a pH from pH 6.8 to pH 8.2.
18 . The composition of claim 5 , wherein the target single-stranded deoxyribonucleic acid lacks a guanine at the 3′ end.
19 . The composition of claim 5 , wherein the terminal 3′ nucleotide in the segment of the target single-stranded deoxyribonucleic acid is A, C or T.
20 . The composition of claim 15 , wherein the Type VI CRISPR-Cas nuclease comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 25.
21 . (canceled)
22 . (canceled)
23 . The composition of claim 1 , wherein the target single-stranded deoxyribonucleic acid has a length of from 18 to 100 nucleotides.
24 . (canceled)
25 . (canceled)
26 . The composition of claim 1 , wherein the composition is comprised within a support medium.
27 . The composition of claim 1 , further comprising a second engineered guide nucleic acid comprising a first segment that is reverse complementary to a segment of a second target deoxyribonucleic acid; and
a Type V CRISPR-Cas nuclease, wherein the second engineered guide nucleic acid comprises a second segment that binds to the Type V CRISPR-Cas nuclease to form a complex.
28 . (canceled)
29 . (canceled)
30 . (canceled)
31 . The composition of claim 1 , wherein the target single-stranded deoxyribonucleic acid is a reverse transcribed ribonucleic acid.
32 . The composition of claim 1 , wherein the composition further comprises a reagent for reverse transcription, amplification, in vitro transcription, or a combination thereof.
33 . (canceled)
34 . (canceled)
35 . The composition of claim 32 , wherein the composition further comprises the reagent for reverse transcription, and wherein the reagent for reverse transcription comprises a reverse transcriptase, an oligonucleotide primer, dNTPs, or any combination thereof.
36 . The composition of claim 32 , wherein the composition further comprises the reagent for amplification, and wherein the reagent for amplification comprises a primer, a polymerase, dNTPs, or any combination thereof.
37 . The composition of claim 32 , wherein the composition further comprises the reagent for in vitro transcription, and wherein the reagent for in vitro transcription comprises an RNA polymerase, NTPs, a primer, or any combination thereof.
38 . A method of assaying for a target single-stranded deoxyribonucleic acid in a sample, the method comprising:
contacting the sample to the composition of any one of claims 1 - 37 ; and
assaying for a signal produced by cleavage of at least some RNA reporters of a plurality of RNA reporters by the Type VI CRISPR-Cas nuclease upon hybridization of the first segment of the engineered guide nucleic acid to the segment of the target single-stranded deoxyribonucleic acid.
39 . A method of assaying for a target ribonucleic acid in a sample, the method comprising:
amplifying the target ribonucleic acid in a sample to produce a target single-stranded deoxyribonucleic acid;
contacting the target single-stranded deoxyribonucleic acid to the composition of any one of claims 1 - 37 ; and
assaying for a signal produced by cleavage of at least some RNA reporters of a plurality of RNA reporters by the Type VI CRISPR-Cas nuclease upon hybridization of the first segment of the engineered guide nucleic acid to the segment of the target single-stranded deoxyribonucleic acid.
40 . (canceled)
41 . (canceled)
42 . (canceled)
43 . A kit comprising:
(a) a Type VI Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas nuclease;
(b) an engineered guide nucleic acid comprising a first segment that is reverse complementary to a segment of a target single-stranded deoxyribonucleic acid; and
(c) a detector nucleic acid comprising a detection moiety.
44 . The kit of claim 43 , further comprising a second engineered guide nucleic acid.
45 . A method of determining the presence of a target single-stranded deoxyribonucleic acid in a sample, the method comprising:
assaying for a signal produced by cleavage of a detection moiety from an RNA reporter, wherein the cleavage occurs when the target single-stranded deoxyribonucleic acid is bound to a complex comprising a Type VI Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas nuclease and an engineered guide nucleic acid comprising a first segment that is reverse complementary to a segment of the target single-stranded deoxyribonucleic acid, wherein if the target single-stranded deoxyribonucleic acid is present in the sample, the signal is detected, thereby determining the presence of the single-stranded deoxyribonucleic acid in the sample.