# US 11,441,137 B2 · CasZ compositions and methods of use

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- Number: US 11,441,137 B2
- Document ID: US11441137B2
- Status: Granted
- Kind: B2
- Application number: 16/884,766
- Filed: 2020-05-27
- Published: 2020-11-26
- Granted: 2022-09-13
- Earliest claimed: 2017-11-01
- Assignees: The Regents of the University of California
- Applicants: The Regents of the University of California
- Inventors: Jennifer A. Doudna; David Burstein; Janice S. Chen; Lucas B. Harrington; David Paez-Espino; Jillian F. Banfield
- Web page: https://patentgrep.com/patent/US11441137B2

## CPC

- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12Q 1/6816: characterised by the detection means (C12Q1/6804 takes precedence)
- C12N 9/1007: {Methyltransferases (general) (2.1.1.)}
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 15/102: {Mutagenizing nucleic acids}
- 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)}
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N 15/86: Viral vectors
- C12N 15/907: {in mammalian cells}
- C12Q 1/6809: Methods for determination or identification of nucleic acids involving differential detection
- C07K 2319/09: containing a nuclear localisation signal
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2750/14143: viral genome or elements thereof as genetic vector
- C12N 2800/80: Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
- 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
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

Provided are compositions and methods that include one or more of: (1) a “CasZ” protein (also referred to as a CasZ polypeptide), a nucleic acid encoding the CasZ protein, and/or a modified host cell comprising the CasZ protein (and/or a nucleic acid encoding the same); (2) a CasZ guide RNA that binds to and provides sequence specificity to the CasZ protein, a nucleic acid encoding the CasZ guide RNA, and/or a modified host cell comprising the CasZ guide RNA (and/or a nucleic acid encoding the same); and (3) a CasZ transactivating noncoding RNA (trancRNA) (referred to herein as a “CasZ trancRNA”), a nucleic acid encoding the CasZ trancRNA, and/or a modified host cell comprising the CasZ trancRNA (and/or a nucleic acid encoding the same).

## Claims

### Claim 1

A method of assaying for a target nucleic acid in a sample, the method comprising:
A) contacting the sample with:
1) a detector nucleic acid; and
2) a composition comprising
a) a polypeptide comprising an amino acid sequence that is at least 95% identical to an amino acid sequence selected from SEQ ID NO:1 and SEQ ID NO:3; and
b) a non-naturally occurring guide nucleic acid, at least a portion of which hybridizes to a segment of the target nucleic acid,
wherein the polypeptide cleaves the detector nucleic acid upon hybridization of the non-naturally occurring guide nucleic acid to the segment of the target nucleic acid; and
B) assaying for a change in a signal,
wherein the change in the signal is produced by cleavage of the detector nucleic acid.

### Claim 2 (depends on 1)

The method of claim 1, wherein the length of the polypeptide is not greater than 550 amino acids.

### Claim 3 (depends on 1)

The method of claim 1, wherein the guide nucleic acid comprises a transactivating noncoding RNA (trancRNA).

### Claim 4 (depends on 1)

The method of claim 1, wherein the detector nucleic acid comprises single stranded DNA.

### Claim 5 (depends on 1)

The method of claim 1, wherein the detector nucleic acid comprises at least one component of a fluorescence-emitting dye pair.

### Claim 6 (depends on 5)

The method of claim 5, wherein the fluorescence-emitting dye pair is selected from a FRET pair; and a quencher and a fluorophore pair.

### Claim 7 (depends on 1)

The method of claim 1, wherein the change in the signal is selected from the group consisting of: a decrease in intensity of the signal, an increase in intensity of the signal, and a change in the wavelength of the signal.

### Claim 8 (depends on 1)

The method of claim 1, wherein the change in the signal persists for at least one minute.

### Claim 9 (depends on 1)

The method of claim 1, wherein the assaying comprises nanoparticle-based detection, fluorescence, fluorescence polarization, colloid phase transition, colloid phase dispersion, electrochemical detection, or any combination thereof.

### Claim 10 (depends on 1)

The method of claim 1, wherein the assaying comprises measuring a detectable signal produced by a reference sample or cell to generate a reference measurement.

### Claim 11 (depends on 1)

The method of claim 1, wherein the polypeptide cleaves single stranded DNA in a non-sequence specific manner upon hybridization of the non-naturally occurring guide nucleic acid to the segment of the target nucleic acid.

### Claim 12 (depends on 1)

The method of claim 1, wherein the detector nucleic acid does not comprise a sequence that is complementary to the portion of the guide nucleic acid that hybridizes to the segment of the target nucleic acid.

### Claim 13 (depends on 1)

The method of claim 1, wherein the target nucleic acid is present in the sample at less than one copy per 1018 non-target DNAs.

### Claim 14 (depends on 1)

The method of claim 1, wherein the target nucleic acid is present in the sample at less than one copy per 1012 non-target DNAs.

### Claim 15 (depends on 1)

The method of claim 1, wherein the target nucleic acid is present in the sample at less than one copy per 107 non-target DNAs.

### Claim 16 (depends on 1)

The method of claim 1, wherein the target nucleic acid is present in the sample at 1 nM or less.

### Claim 17 (depends on 1)

The method of claim 1, wherein the target nucleic acid is present in the sample at 100 fM or less.

### Claim 18 (depends on 1)

The method of claim 1, wherein the target nucleic acid is single stranded DNA.

### Claim 19 (depends on 1)

The method of claim 1, wherein the target nucleic acid is a bacterial nucleic acid.

### Claim 20 (depends on 1)

The method of claim 1, wherein the sample comprises a cell lysate.

### Claim 21 (depends on 1)

The method of claim 1, wherein the reference sample comprises a positive control target nucleic acid.

### Claim 22 (depends on 1)

The method of claim 1, wherein the reference sample comprises a control non-target nucleic acid.

### Claim 23 (depends on 1)

The method of claim 1, wherein the polypeptide has an amino acid sequence identity of at least 98% to SEQ ID NO:1 or SEQ ID NO:3.

### Claim 24 (depends on 1)

The method of claim 1, wherein the target nucleic acid is a eukaryotic nucleic acid.

### Claim 25 (depends on 1)

The method of claim 1, wherein the target nucleic acid is viral nucleic acid.

### Claim 26 (depends on 1)

The method of claim 1, wherein the amino acid sequence of the polypeptide is at least 99% identical to SEQ ID NO: 1 or SEQ ID NO: 3.

### Claim 27 (depends on 1)

The method of claim 1, wherein the target nucleic acid is double stranded DNA.

### Claim 28 (depends on 1)

The method of claim 1, wherein the guide nucleic acid comprises a transactivating CRISPR RNA (tracrRNA).

### Claim 29 (depends on 1)

The method of claim 1, wherein the detector nucleic acid consists of single stranded DNA.