# US 2023/0053573 A1 · Nucleic acid detection using type iii crispr complex

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- Number: US 2023/0053573 A1
- Document ID: US20230053573A1
- Status: Published application
- Kind: A1
- Application number: 17/814,097
- Filed: 2022-07-21
- Published: 2023-02-23
- Earliest claimed: 2021-07-21
- Assignees: Montana State University
- Applicants: Montana State University
- Inventors: Blake A. WIEDENHEFT; Andrew SANTIAGO-FRANGOS; Anna A. NEMUDRAIA; Artem A. NEMUDRYI
- Web page: https://patentgrep.com/patent/US20230053573A1

## CPC

- C12Q 1/70: involving virus or bacteriophage {(immunoassay for viruses G01N33/56983)}
- C12Q 1/701: {Specific hybridization probes}
- C12Q 1/682: Signal amplification
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6818: involving interaction of two or more labels, e.g. resonant energy transfer
- C12Q 1/6853: using modified primers or templates
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

The disclosure relates to engineered systems and methods for detecting target nucleic acid in a sample, which may be a complex mixture. The systems and methods may improve sensitivity of target nucleic acid detection by enhancing signal generation. For example, signal generation may be enhanced through programmable capture and concentration of the target nucleic acid using an engineered type III CRISPR complex. Various ancillary nucleases such as Can1, Can2, and NucC are identified and may be used for detection. For example, binding of the engineered type III CRISPR complex may produce products that activate the identified ancillary nucleases. Different activators trigger changes in the substrate specificity of these nucleases. The activated nucleases may be used to detect programmatic detection of the target nucleic in the sample. The systems and methods are shown to detect viral RNA directly from nasopharyngeal swab samples.

## Claims

### Claim 1

A method of programmatically capturing and detecting target nucleic acid in a sample based on an engineered type III Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) complex, the method comprising:
capturing the target nucleic acid in the sample using the engineered type III CRISPR-Cas complex, the engineered type III CRISPR-Cas complex comprising: a CRISPR guide comprising a CRISPR guide sequence engineered to be complementary to the target nucleic acid;
concentrating the captured target nucleic acid to amplify detectable activity of the engineered type III CRISPR-Cas complex; and
detecting the captured and concentrated target nucleic acid based on the detectable activity of the engineered type III CRISPR-Cas complex.

### Claim 2 (depends on 1)

The method of claim 1, wherein the sample comprises a complex mixture and wherein the capturing and concentrating are performed without prior amplification of the target nucleic acid in the complex mixture.

### Claim 3 (depends on 1)

The method of claim 1, wherein the detectable activity comprises generation of a linear or cyclic oligonucleotide.

### Claim 4 (depends on 3)

The method of claim 3, wherein the generation of the linear or cyclic oligonucleotide is detectable via a nuclease that is activated by the linear or cyclic oligonucleotide.

### Claim 5 (depends on 4)

The method of claim 4, wherein the nuclease comprises a Can nuclease and wherein the type III CRISPR system comprises the Can nuclease.

### Claim 6 (depends on 5)

The method of claim 5, wherein the Can nuclease comprises Can1 and/or Can2.

### Claim 7 (depends on 4)

The method of claim 4, wherein the nuclease comprises a NucC nuclease and wherein the type III CRISPR system comprises the NucC nuclease.

### Claim 8 (depends on 7)

The method of claim 7, wherein contacting the nucleic acid in the sample with the engineered type III CRISPR-Cas complex comprises:
contacting the captured and concentrated nucleic acid with a fluorophore and a quencher tethered together by a nucleic acid tether, wherein the activated nuclease cleaves the nucleic acid tether to thereby release the fluorophore from the quencher; and
detecting a level of fluorescence of the released fluorophore.

### Claim 9 (depends on 8)

The method of claim 8, wherein the tether comprises a ribonucleic acid and/or a deoxyribonucleic acid tether.

### Claim 10 (depends on 1)

The method of claim 1, wherein the engineered type III CRISPR complex is Histidine-tagged (His-tagged), and wherein capturing the target nucleic acid comprises:
incubating the sample with the His-tagged engineered type III CRISPR complex to bind the His-tagged engineered type III CRISPR complex with the target nucleic acid; and
binding the His-tagged engineered type III CRISPR complex with bound target nucleic acid to magnetic particles.

### Claim 11 (depends on 10)

The method of claim 10, wherein the magnetic particles comprise Ni-NTA beads.

### Claim 12 (depends on 10)

The method of claim 10, wherein concentrating the captured target nucleic acid comprises:
applying a magnet to generate a pellet comprising the His-tagged engineered type III CRISPR complex bound to the target nucleic acid and the magnetic particles.

### Claim 13 (depends on 12)

The method of claim 12, wherein the detectable activity comprises generation of a linear or cyclic oligonucleotide that is detectable via a nuclease activated by the linear or cyclic oligonucleotide, the method further comprising:
resuspending the pellet in a solution comprising reaction buffer containing ATP to activate polymerase activity of the His-tagged engineered type III CRISPR complex, wherein the polymerase activity generates the linear or cyclic oligonucleotide that activates the nuclease.

### Claim 14 (depends on 13)

The method of claim 13, further comprising:
adding the nuclease, a fluorophore, and a quencher tethered together by a nucleic acid tether, wherein the activated nuclease cleaves the nucleic acid tether to release the fluorophore; and
detecting a level of fluorescence of the released fluorophore.

### Claim 15 (depends on 14)

The method of claim 14, wherein the linear or cyclic oligonucleotide comprises cA3 and/or cA4 that activates a NucC nuclease.

### Claim 16 (depends on 14)

The method of claim 14, wherein the linear or cyclic oligonucleotide comprises cA3 and/or cA4 that activates Can nuclease.

### Claim 17 (depends on 1)

The method of claim 1, wherein the sample is a complex mixture obtained from a subject.

### Claim 18 (depends on 17)

The method of claim 17, wherein the sample comprises a nasopharyngeal swab sample.

### Claim 19 (depends on 1)

The method of claim 1, wherein the target nucleic acid in the sample comprises ribonucleic acid (RNA).

### Claim 20 (depends on 19)

The method of claim 19, wherein the RNA comprises at least a portion of an RNA genome of a virus.

### Claim 21 (depends on 20)

The method of claim 20, wherein the viral RNA comprises an RNA from the genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

### Claim 22 (depends on 1)

The method of claim 1, wherein the CRISPR guide further comprises a 3′ protospacer flanking sequence that activates or enhances generation of linear or cyclic oligonucleotides.