CRISPR-BASED SARS-COV-2 DETECTION

1 . A composition comprising

(i) a guide polynucleotide having a nucleotide sequence comprising a crRNA selected from the group consisting of: SEQ ID NO.: 77; SEQ ID NO.: 84; SEQ ID NO.: 91; and

(ii) a CRISPR/Cas enzyme.

2 . The composition of claim 1 , wherein the CRISPR/Cas enzyme is a Type V CRISPR/Cas enzyme.

3 . The composition of claim 1 , wherein the CRISPR/Cas enzyme is a Type VI CRISPR/Cas enzyme.

4 . The composition of claim 1 , wherein the CRISPR/Cas enzyme is a thermostable CRISPR/Cas enzyme.

5 . The composition of claim 1 , wherein the CRISPR/Cas enzyme is a Cas13 CRISPR/Cas enzyme.

6 . A composition comprising:

a reverse transcriptase;

a DNA polymerase;

nucleotides;

RNA;

a primer set selected from the group consisting of:

(a):

orf1ab-F3

(SEQ ID NO. 72)

TGAAAATAGGACCTGAGCG

orf1ab-B3

(SEQ ID NO. 73)

ACACCTAGTCATGATTGCA

orf1ab-FIP

(SEQ ID NO. 74)

CCAATAGAATGATGCCAACAGGCGATAGACGTGCCACATGC

orf1ab-BIP

(SEQ ID NO. 75)

GATTGATGTTCAACAATGGGGTTTCATTACCATGGACTTGACAAT

orf1ab-LF-T7

(SEQ ID NO. 76)

gaaatTAATACGACTCACTATAGGGAAGTGTCTGAAGCAGTGGAAAA;

(b):

N-5F3

(SEQ ID NO. 78)

GCTTCTACGCAGAAGGGA

N-5B3 

(SEQ ID NO. 79)

GTGACAGTTTGGCCTTGT

N-5FIP

(SEQ ID NO. 80)

TACTGCTGCCTGGAGTTGAATTCCTCTTCTCGTTCCTCATC

N-5BIP

(SEQ ID NO. 81)

GCTTTGCTGCTGCTTGACAGTGTTGTTGGCCTTTACCA

N-5LB

(SEQ ID NO. 82)

ATTGAACCAGCTTGAGAGCAAA

N-5LF-T7

(SEQ ID NO. 83)

gaaatTAATACGACTCACTATAGGGCTTGAACTGTTGCGACTACGT;

(c)

RP-F3

(SEQ ID NO. 85)

TTGATGAGCTGGAGCCA

RP-B3 

(SEQ ID NO. 86)

CACCCTCAATGCAGAGTC

RP-FIP

(SEQ ID NO. 87)

GTGTGACCCTGAAGACTCGGTTTTAGCCACTGACTCGGATC

RP-BIP

(SEQ ID NO. 88)

CCTCCGTGATATGGCTCTTCGTTTTTTTCTTACATGGCTCTGGTC

RP-LF

(SEQ ID NO. 89)

ATGTGGATGGCTGAGTTGTT

RP-LB-T7

(SEQ ID NO. 90)

GAATTAATACGACTCACTATAGGGCATGCTGAGTACTGGACCTC;

and combinations thereof.

7 . The composition of claim 6 , wherein the RNA is isolated from a sample from a subject.

8 . The composition of claim 7 , wherein the subject is or is suspected to be or have been exposed to or infected with SARS-CoV-2.

9 .- 18 . (canceled)

19 . A method of detecting a SARS-CoV-2 target nucleic acid sequence, the method comprising:

contacting an RNA preparation with a CRISPR/Cas enzyme bound to a guide polynucleotide having a nucleotide sequence comprising a crRNA selected from the group consisting of: SEQ ID NO.: 77; SEQ ID NO.: 84; SEQ ID NO.: 91;

in the presence of rNTPs and a labeled nucleic acid reporter construct;

wherein cleavage of the labeled nucleic acid reporter construct by the CRISPR/Cas enzyme results in a detectable signal;

wherein detection of the detectable signal indicates presence of the SARS-CoV-2 target nucleic acid sequence in the RNA preparation.

20 . A method of detecting a SARS-CoV-2 target nucleic acid sequence, the method comprising:

(i) obtaining a sample from a subject

(ii) isolating nucleic acid from the sample

(iii) amplifying target sequences by contacting the isolated nucleic acid with a primer set selected from the group consisting of:

(a):

orf1ab-F3

(SEQ ID NO. 72)

TGAAAATAGGACCTGAGCG

orf1ab-B3

(SEQ ID NO. 73)

ACACCTAGTCATGATTGCA

orf1ab-FIP

(SEQ ID NO. 74)

CCAATAGAATGATGCCAACAGGCGATAGACGTGCCACATGC

orf1ab-BIP

(SEQ ID NO. 75)

GATTGATGTTCAACAATGGGGTTTCATTACCATGGACTTGACAAT

orf1ab-LF-T7

(SEQ ID NO. 76)

gaaatTAATACGACTCACTATAGGGAAGTGTCTGAAGCAGTGGAAAA;

(b):

N-5F3

(SEQ ID NO. 78)

GCTTCTACGCAGAAGGGA

N-5B3

(SEQ ID NO. 79)

GTGACAGTTTGGCCTTGT

N-5FIP

(SEQ ID NO. 80)

TACTGCTGCCTGGAGTTGAATTCCTCTTCTCGTTCCTCATC

N-5BIP

(SEQ ID NO. 81)

GCTTTGCTGCTGCTTGACAGTGTTGTTGGCCTTTACCA

N-5LB

(SEQ ID NO. 82)

ATTGAACCAGCTTGAGAGCAAA

N-5LF-T7

(SEQ ID NO. 83)

gaaatTAATACGACTCACTATAGGGCTTGAACTGTTGCGACTACGT;

(c)

RP-F3

(SEQ ID NO. 85)

TTGATGAGCTGGAGCCA

RP-B3

(SEQ ID NO. 86)

CACCCTCAATGCAGAGTC

RP-FIP

(SEQ ID NO. 87)

GTGTGACCCTGAAGACTCGGTTTTAGCCACTGACTCGGATC

RP-BIP

(SEQ ID NO. 88)

CCTCCGTGATATGGCTCTTCGTTTTTTTCTTACATGGCTCTGGTC

RP-LF

(SEQ ID NO. 89)

ATGTGGATGGCTGAGTTGTT

RP-LB-T7

(SEQ ID NO. 90)

GAATTAATACGACTCACTATAGGGCATGCTGAGTACTGGACCTC;

and combinations thereof.

a reverse transcriptase;

a DNA polymerase;

nucleotides

(iv) contacting the amplified target sequences with:

a CRISPR/Cas enzyme;

a guide polynucleotide having a nucleotide sequence comprising a crRNA selected from the group consisting of: SEQ ID NO.: 77; SEQ ID NO.: 84; SEQ ID NO.: 91

rNTPs;

a labeled nucleic acid reporter construct;

wherein cleavage of the labeled nucleic acid reporter construct by the CRISPR/Cas enzyme results in a detectable signal;

wherein detection of the detectable signal indicates presence of the SARS-CoV-2 target nucleic acid sequence.

21 . (canceled)

22 . The method of claim 20 , wherein the sample is a biological sample.

23 . The method of claim 20 , wherein the sample comprises nasal swab, nasopharyngeal swab, oropharyngeal swab, nasal aspirate, sputum, bronchoalveolar lavage, blood, serum, feces, and saliva.

24 . The method of claim 20 , wherein the step of isolating nucleic acid from the sample comprises isolating RNA from the sample.

25 . The method of claim 20 , wherein the labeled nucleic acid reporter construct is an RNA.

26 . The method of claim 20 , wherein the labeled nucleic acid reporter construct comprises a fluor/quencher pair.

27 . The method of claim 20 , wherein the detectable signal is fluorescence detection.

28 . The composition of claim 20 , wherein the CRISPR/Cas enzyme is a Type V CRISPR/Cas enzyme.

29 . The composition of claim 20 , wherein the CRISPR/Cas enzyme is a Type VI CRISPR/Cas enzyme.

30 . The composition of claim 20 , wherein the CRISPR/Cas enzyme is a thermostable CRISPR/Cas enzyme.

31 . The composition of claim 20 , wherein the CRISPR/Cas enzyme is a Cas13 CRISPR/Cas enzyme.

32 . The composition of claim 20 , wherein the CRISPR/Cas enzyme exhibits collateral RNase activity.

33 .- 41 . (canceled)

42 . A method of detecting a SARS-CoV-2 target nucleic acid sequence in a sample comprising:

contacting nucleic acid from the sample with:

a primer set having at least one primer selected from the group consisting of SEQ ID NOs.: 72-76, 78-83 92-429, 528-529 and JpS1-FIP AGGGACATAAGTCACATGCAAGAAGCTATCATCTTATGTCCTTCCCTC;

a type VI Cas;

at least one guide polynucleotide comprising a crRNA capable of binding the target nucleic acid sequence; and

a labeled nucleic acid reported construct, wherein the type VI Cas exhibits collateral RNase activity and cleaves the labeled nucleic acid reported construct once activated by presence of the target sequence;

detecting a signal from cleavage of labeled nucleic acid reported construct, thereby detecting the SARS-CoV-2 target nucleic acid sequence in the sample.

43 . The method of claim 42 , wherein the type VI Cas is Cas13.

44 . The method of claim 42 , further comprising obtaining a biological sample from a subject.

45 . The method of claim 42 , wherein the biological sample comprises nasal swab, nasopharyngeal swab, oropharyngeal swab, nasal aspirate, sputum, bronchoalveolar lavage, blood, feces, and saliva samples.

46 . The method of claim 42 , wherein the step of detecting a signal comprises detection of fluorescence, absorbance, spectrometry, lateral flow, migration, chemiluminescence, migration, electrochemical detection.

47 .- 60 . (canceled)