# US 10,370,700 B2 · Detection of targeted sequence regions

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- Number: US 10,370,700 B2
- Document ID: US10370700B2
- Status: Granted
- Kind: B2
- Application number: 16/132,955
- Filed: 2018-09-17
- Published: 2019-01-24
- Granted: 2019-08-06
- Earliest claimed: 2017-06-13
- Assignees: Genetics Research, LLC
- Applicants: Genetics Research, LLC
- Inventors: Anthony P. Shuber; William Glover; Thomas C. Meyers
- Web page: https://patentgrep.com/patent/US10370700B2

## CPC

- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6869: Methods for sequencing
- C12Q 1/701: {Specific hybridization probes}
- 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 herein are methods of detecting nucleic acids. The nucleic acid of interest may be detected by using Cas endonuclease to degrade substantially all nucleic acid in a sample except for the nucleic acid of interest, leaving the nucleic acid of interest isolated and amenable to detection. In related methods, Cas endonuclease complexes are used to protect the nucleic acid of interest while unprotected nucleic acid is digested, e.g., by exonuclease, after which the isolated nucleic acid of interest is detected.

## Claims

### Claim 1

A method of detecting a nucleic acid, the method comprising:
protecting, in a population of nucleic acids in a sample from an organism, first and second ends of a target nucleic acid using a first Cas endonuclease and a second Cas endonuclease, wherein the target nucleic acid is endogenous to the organism;
digesting unprotected nucleic acids with exonuclease; and
detecting the protected target nucleic acid.

### Claim 2 (depends on 1)

The method of claim 1, wherein:
the first Cas endonuclease and the second Cas endonuclease complex are different,
substantially all of the unprotected nucleic acids are degraded, and
the target nucleic acid is from a genome of the organism.

### Claim 3 (depends on 1)

The method of claim 1, wherein the organism is a human.

### Claim 4 (depends on 1)

The method of claim 1, wherein the first Cas endonuclease comprises a first Cas9 protein complexed with a first guide RNA, and the second Cas endonuclease comprises a second Cas9 protein complexed with a second guide RNA.

### Claim 5 (depends on 4)

The method of claim 4, wherein at least one of the first Cas9 complex and the second Cas9 complex comprises a catalytically inactive Cas9 protein.

### Claim 6 (depends on 1)

The method of claim 1, wherein the population of nucleic acids is isolated from the organism and the target nucleic acid comprises a sequence from a genome of the organism.

### Claim 7 (depends on 1)

The method of claim 1, wherein: the population of nucleic acids is isolated from an organism and the target nucleic acid is from a mitochondrial genome of the organism.

### Claim 8 (depends on 1)

The method of claim 1, wherein the detecting step comprises one selected from the group consisting of hybridization, spectrophotometry, sequencing, electrophoresis, amplification, fluorescence detection, and chromatography.

### Claim 9 (depends on 1)

The method of claim 1, wherein the detecting step comprises sequencing.

### Claim 10 (depends on 1)

The method of claim 1, wherein the sample comprises at least one selected from the group consisting of blood, serum, and plasma.

### Claim 11

A method of analyzing nucleic acid, the method comprising:
protecting, in a population of nucleic acids, first and second ends of a target nucleic acid using a first Cas endonuclease and a second Cas endonuclease;
digesting unprotected nucleic acids with exonuclease while the target nucleic acid is protected by the first Cas endonuclease and a second Cas endonuclease; and
sequencing the target nucleic acid.

### Claim 12 (depends on 11)

The method of claim 11, wherein the population of nucleic acids is isolated from an organism and the target nucleic acid comprises a sequence foreign to a genome of the organism.

### Claim 13 (depends on 12)

The method of claim 12, wherein the target nucleic acid is from a genome of a pathogen of the organism.