# US 12,215,377 B2 · HIV or HCV detection with CRISPR-CAS13A

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- Number: US 12,215,377 B2
- Document ID: US12215377B2
- Status: Granted
- Kind: B2
- Application number: 17/273,752
- Filed: 2019-09-06
- Published: 2021-11-11
- Granted: 2025-02-04
- Earliest claimed: 2018-09-07
- Assignees: The J. David Gladstone Institute, a testamentary trust established under the Will of J. David Gladstone; The Regents of the University of California
- Applicants: The Regents of the University of California; The J. David Gladstone Institutes, a testamentary trust established under the Will of J. David Gladstone
- Inventors: Parinaz Fozouni; Melanie Ott; Jennifer A. Doudna
- Web page: https://patentgrep.com/patent/US12215377B2

## CPC

- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 15/11: DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity}
- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- C12Q 1/6888: for detection or identification of organisms
- C12Q 1/703: {Viruses associated with AIDS}
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12Q 2600/156: Polymorphic or mutational markers
- C12Q 2600/158: Expression markers
- 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present disclosure relates to methods using CRISPR-Cas13a enzyme, complexed with HIV or HCV crRNAs to specifically and sensitively detect and quantify the presence of HIV or HCV RNA in a sample. These methods can be used to diagnose HIV or HCV infection, quantify the concentration of HIV or HCV RNA present in a sample, identify the presence of different HIV or HCV splice variants, subtypes, or mutations, and to monitor reactivation of HIV or HCV transcription.

## Claims

### Claim 1

A method comprising:
(a) incubating a sample containing RNA with a Cas13a protein and at least one CRISPR guide RNA (crRNA) for a period of time to form a RNA cleavage product; and
(b) detecting a level of HIV or HCV RNA cleavage product with a detector,
wherein the RNA is not reverse transcribed prior to the detecting step,
wherein the least one crRNA is SEQ ID NOs: 1-8, 18-25 or 26.

### Claim 2 (depends on 1)

The method of claim 1, further comprising a step of amplification of the RNA and/or the HIV or HCV RNA cleavage products.

### Claim 3 (depends on 1)

The method of claim 1, wherein the RNA and/or the HIV or HCV RNA cleavage product are not amplified.

### Claim 4 (depends on 1)

The method of claim 1, wherein the detector is a fluorescence detector, optionally a short quenched-fluorescent RNA.

### Claim 5 (depends on 1)

The method of claim 1, wherein the sample is incubated with 2, 3, 4, 5, 6, 7, 8, 9, 10 or more crRNAs.

### Claim 6 (depends on 1)

The method of claim 1, wherein the HIV or HCV RNA cleavage product concentration is determined using a standard curve and the level of the HIV or HCV RNA cleavage product.

### Claim 7 (depends on 1)

The method of claim 1, further comprising depleting a portion of the sample prior to detecting step.

### Claim 8 (depends on 7)

The method of claim 7, wherein the portion of the sample is a human nucleic acid portion.

### Claim 9 (depends on 1)

The method of claim 1, further comprising treating the sample with an RNase inhibitor and/or heat before the incubating step.

### Claim 10 (depends on 1)

The method of claim 1, wherein the Cas13a protein and/or crRNA is lyophilized prior to incubation with the sample.

### Claim 11

A method for quantifying HIV or HCV RNA concentration comprising:
(a) incubating a sample containing RNA with a Cas13a protein and at least one CRISPR guide RNA (crRNA) for a period of time; and
(b) analyzing the sample for HIV or HCV RNA cleavage product concentration with a detector,
wherein the RNA is not reverse transcribed prior to the analyzing step,
wherein the least one crRNA is SEQ ID NOs: 1-8, 18-25 or 26.

### Claim 12 (depends on 11)

The method of claim 11, wherein the HIV or HCV RNA cleavage product concentration is determined using a standard curve.

### Claim 13 (depends on 11)

The method of claim 11, further comprising a step of amplification of the RNA and/or the HIV or HCV RNA cleavage product.

### Claim 14 (depends on 11)

The method of claim 11, wherein the detector is a fluorescence detector, optionally a short quenched-fluorescent RNA.

### Claim 15 (depends on 11)

The method of claim 11, wherein the sample is incubated with 2, 3, 4, 5, 6, 7, 8, 9, 10 or more crRNAs.

### Claim 16 (depends on 11)

The method of claim 11, further comprising depleting a portion of the sample prior to the analyzing step.

### Claim 17 (depends on 11)

The method of claim 11, further comprising removing RNase from the sample before the incubating step.

### Claim 18 (depends on 17)

The method of claim 17, further comprising treating the sample with an RNase inhibitor and/or heat before the incubating step.

### Claim 19 (depends on 11)

The method of claim 11, wherein the Cas13a protein and/or crRNA is lyophilized prior to incubation with the sample.