# US 2020/0399697 A1 · Type v crispr/cas effector proteins for cleaving ssdnas and detecting target dnas

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- Number: US 2020/0399697 A1
- Document ID: US20200399697A1
- Status: Published application
- Kind: A1
- Application number: 16/927,351
- Filed: 2020-07-13
- Published: 2020-12-24
- Earliest claimed: 2017-11-22
- Assignees: The Regents of the University of California
- Applicants: The Regents of the University of California
- Inventors: Jennifer A. Doudna; Janice S. Chen; Lucas Benjamin Harrington; Enbo Ma
- Web page: https://patentgrep.com/patent/US20200399697A1

## CPC

- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12N 9/222: {Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes}
- 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}
- C12Q 1/6823: Release of bound markers
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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

Provided are compositions and methods for detecting a target DNA (double stranded or single stranded) in a sample. In some embodiments, a subject method includes: (a) contacting the sample with: (i) a type V CRISPR/Cas effector protein (e.g., a Cas12 protein such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e); (ii) a guide RNA (comprising a region that binds to the type V CRISPR/Cas effector protein, and a guide sequence that hybridizes with the target DNA); and (iii) a detector DNA that is single stranded (i.e., a “single stranded detector DNA”) and does not hybridize with the guide sequence of the guide RNA; and (b) measuring a detectable signal produced by cleavage (by the type V CRISPR/Cas effector protein) of the single stranded detector DNA. Also provided are compositions and methods for cleaving single stranded DNAs (e.g., non-target ssDNAs), e.g., inside of a cell.

## Claims

### Claim at position 1

-45. (canceled)

### Claim 46

A method of multiplexed detection of target nucleic acids in a sample, the method comprising:
a) contacting the sample to:
i) an RNA-guided nuclease comprising a RuvC domain;
ii) a first guide nucleic acid that hybridizes to a segment of a first target nucleic acid;
iii) a second guide nucleic acid that hybridizes to a segment of a second target nucleic acid; and
iv) a detector nucleic acid; and
b) assaying for a signal produced by cleavage of the detector nucleic acid by the RNA-guided nuclease.

### Claim 47 (depends on 46)

The method of claim 46, further comprising contacting the sample to one or more additional guide nucleic acids that hybridize to a segment of one or more additional target nucleic acids.

### Claim 48 (depends on 46)

The method of claim 46, wherein the first target nucleic acid, the second target nucleic acid, or both, are single stranded.

### Claim 49 (depends on 46)

The method of claim 46, wherein the first target nucleic acid, the second target nucleic acid, or both, are double stranded.

### Claim 50 (depends on 46)

The method of claim 46, wherein the first target nucleic acid, the second target nucleic acid, or both, is present in the sample at a concentration as low as 200 fM.

### Claim 51 (depends on 46)

The method of claim 46, wherein the first target nucleic acid, the second target nucleic acid, or both, is present in the sample at one or more copies per 107 non-target nucleic acids.

### Claim 52 (depends on 46)

The method of claim 46, wherein the RNA-guided nuclease exhibits trans cleavage activity subsequent to binding to the first target nucleic acid or the second target nucleic acid.

### Claim 53 (depends on 46)

The method of claim 46, wherein the first target nucleic acid, the second target nucleic acid, or both, are associated with a disease state.

### Claim 54 (depends on 46)

The method of claim 46, wherein the first target nucleic acid, the second target nucleic acid, or both, are viral nucleic acids or bacterial nucleic acids.

### Claim 55 (depends on 54)

The method of claim 54, wherein the viral nucleic acids are papovavirus, human papillomavirus (HPV), hepadnavirus, Hepatitis B Virus (HBV), herpesvirus, varicella zoster virus (VZV), Epstein Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus, adenovirus, poxvirus, or parvovirus, an influenza virus, or a respiratory syncytial virus nucleic acid.

### Claim 56 (depends on 46)

The method of claim 46, wherein the first target nucleic acid is a nucleic acid from a first viral strain or a first bacterial strain and the second target nucleic acid is a nucleic acid from a second viral strain or a second bacterial strain.

### Claim 57 (depends on 46)

The method of claim 46, wherein the first target nucleic acid is a nucleic acid from a first viral strain or a first bacterial strain and the second target nucleic acid is a nucleic acid from the first viral strain or the first bacterial strain.

### Claim 58 (depends on 46)

The method of claim 46, wherein the first target nucleic acid and the second target nucleic acid are the same.

### Claim 59 (depends on 56)

The method of claim 56, wherein the first viral strain is a first strain of influenza and the second viral strain is a second strain of influenza.

### Claim 60 (depends on 56)

The method of claim 56, wherein the first viral strain is a first strain of influenza virus and the second viral strain is a second strain of respiratory syncytial virus.

### Claim 61 (depends on 46)

The method of claim 46, wherein the first target nucleic acid, the second target nucleic acid, or both, comprise a single nucleotide polymorphism.

### Claim 62 (depends on 46)

The method of claim 46, wherein the first target nucleic acid, the second target nucleic acid, or both, are from a human cell.

### Claim 63 (depends on 46)

The method of claim 46, wherein the first target nucleic acid, the second target nucleic acid, or both are human fetal nucleic acids, cancer cell nucleic acids, or a combination thereof.

### Claim 64 (depends on 46)

The method of claim 46, wherein the RNA-guided nuclease comprises a single catalytic domain comprising the RuvC domain.

### Claim 65 (depends on 46)

The method of claim 46, wherein the RNA-guided nuclease is a Cas12 protein.

### Claim 66 (depends on 46)

The method of claim 46, wherein the RNA-guided nuclease is a Cas12a polypeptide, a Cas12b polypeptide, a Cas12c polypeptide, a Cas12d polypeptide, a Cas12e polypeptide, a C2c4 polypeptide, a C2c8 polypeptide, a C2c5 polypeptide, a C2c10 polypeptide, and a C2c9 polypeptide.

### Claim 67 (depends on 46)

The method of claim 46, wherein the sample comprises a cell or a cell lysate.

### Claim 68 (depends on 46)

The method of claim 46, wherein the sample is blood, saliva, biopsy, plasma, serum, bronchoalveolar lavage, sputum, a fecal sample, cerebrospinal fluid, a fine needle aspirate, a buccal swab, a cervical swab, a nasal swab, interstitial fluid, synovial fluid, nasal discharge, tears, buffy coat, or a mucous membrane sample.

### Claim 69 (depends on 46)

The method of claim 46, comprising determining an amount of the first target nucleic acid and an amount of the second target nucleic acid present in the sample.

### Claim 70 (depends on 46)

The method of claim 46, wherein the assaying for the signal produced by cleavage of the detector nucleic acid 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, and semiconductor-based sensing.

### Claim 71 (depends on 46)

The method of claim 46, wherein the detector nucleic acid produces the signal prior to cleavage of the detector nucleic acid, and the signal is reduced after cleavage of the detector nucleic acid.

### Claim 72 (depends on 46)

The method of claim 46, wherein the signal increases after cleavage of the detector nucleic acid.

### Claim 73 (depends on 46)

The method of claim 46, wherein the detector nucleic acid comprises one or more fluorescence-emitting dye pairs.

### Claim 74 (depends on 73)

The method of claim 73, wherein the one or more fluorescence-emitting dye pairs includes a fluorescence resonance energy transfer (FRET) pair, a quencher and fluorophore pair, or a combination thereof.

### Claim 75

A kit for multiplexed detection of target nucleic acids in a sample, the kit comprising:
(a) a first guide nucleic acid and a second guide nucleic acid;
wherein the first guide nucleic acid comprises a region that binds to an RNA-guided nuclease comprising a RuvC domain, and a first guide sequence that hybridizes to a segment of a first target nucleic acid;
wherein the second guide nucleic acid comprises a region that binds to the RNA-guided nuclease, and a second guide sequence that hybridizes to a segment of a second target nucleic acid; and
(b) a detector nucleic acid that produces a detectable signal upon cleavage of the detector nucleic acid by the RNA-guided nuclease.