# US 10,501,728 B2 · Engineered nucleic-acid targeting nucleic acids

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- Number: US 10,501,728 B2
- Document ID: US10501728B2
- Status: Granted
- Kind: B2
- Application number: 16/224,747
- Filed: 2018-12-18
- Published: 2019-04-18
- Granted: 2019-12-10
- Earliest claimed: 2015-10-23
- Assignees: Caribou Biosciences, Inc.
- Applicants: Caribou Biosciences, Inc.
- Inventors: Paul Daniel Donohoue; Andrew Paul May
- Web page: https://patentgrep.com/patent/US10501728B2

## CPC

- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 9/96: Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
- C12N 15/102: {Mutagenizing nucleic acids}
- 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 15/902: {using homologous recombination}
- C12N 15/907: {in mammalian cells}
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2310/3519: Fusion with another nucleic acid
- C12N 2310/51: in polymeric form, e.g. multimers, concatemers
- 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 provides engineered cross-type-nucleic-acid targeting nucleic acids and compositions thereof. Nucleic acid sequences encoding the engineered cross-type-nucleic-acid targeting nucleic acids, as well as expression cassettes, vectors and cells comprising such nucleic acid sequences, are described. Also, methods are disclosed for making and using the engineered cross-type-nucleic-acid targeting nucleic acids and compositions thereof.

## Claims

### Claim 1

An engineered CRISPR Class 2 cross-type-nucleic-acid targeting nucleic acid (“CRISPR Class 2 cross-type-NATNA”), comprising:
a first CRISPR Type II Cas9-associated nucleic-acid targeting nucleic acid, having a 5′ end and a 3′ end, comprising a spacer element complementary to a first nucleic acid target sequence (“first Cas9-NATNA”);
a second CRISPR Type II Cas9-associated nucleic-acid targeting nucleic acid, having a 5′ end and a 3′ end, comprising a tracr element (“second Cas9-NATNA”) and a first linker element, having a 5′ end and a 3′ end, wherein the 3′ end of the first linker element is covalently connected at the 5′ end of the second Cas9-NATNA;
a first CRISPR Type V Cpf1-associated nucleic-acid targeting nucleic acid, having a 5′ end and a 3′ end, comprising a spacer element complementary to a second nucleic acid target sequence (“first Cpf1-NATNA”), wherein the 3′ end is covalently connected with the 5′ end of the first linker element; and
a second linker element, having a 5′ and a 3′ end, wherein the second linker element is covalently connected at the 5′ end of the first Cas9-NATNA or at the 3′ end of the first Cas9-NATNA or at the 3′ end of the second Cas9-NATNA.

### Claim 2 (depends on 1)

The engineered CRISPR Class 2 cross-type-NATNA of claim 1, wherein the first Cpf1-NATNA is capable of forming a complex with a CRISPR Type V Cpf1 protein (“CRISPR Class 2 cross-type-NATNA/Cpf1 protein complex”), and wherein the CRISPR Class 2 cross-type-NATNA/Cpf1 protein complex is capable of binding the second nucleic acid target sequence.

### Claim 3 (depends on 2)

The engineered CRISPR Class 2 cross-type-NATNA of claim 2, wherein the first Cas9-NATNA and the second Cas9-NATNA are capable of forming a complex with a CRISPR Type II Cas9 protein (“CRISPR Class 2 cross-type-NATNA/Cpf1 protein/Cas9 protein complex”), and wherein the CRISPR Class 2 cross-type-NATNA/Cpf1 protein/Cas9 protein complex is capable of binding the first nucleic acid target sequence and the second nucleic acid target sequence.

### Claim 4 (depends on 1)

The engineered CRISPR Class 2 cross-type-NATNA of claim 1, wherein at least one of the first Cpf1-NATNA, the first Cas9-NATNA, or the second Cas9-NATNA comprises RNA.

### Claim 5 (depends on 1)

The engineered CRISPR Class 2 cross-type-NATNA of claim 1, wherein at least one of the first Cpf1-NATNA, the first Cas9-NATNA, or the second Cas9-NATNA comprises DNA.

### Claim 6 (depends on 1)

A nucleic acid/protein composition, comprising:
the engineered CRISPR Class 2 cross-type-NATNA of claim 1;
a catalytically active or catalytically inactive CRISPR Type II Cas9 protein; and
a catalytically active or catalytically inactive CRISPR Type V Cpf1 protein.

### Claim 7 (depends on 6)

The nucleic acid/protein composition of claim 6, wherein the engineered CRISPR Class 2 cross-type-NATNA is in a complex with the CRISPR Type II Cas9 protein and the CRISPR Type V Cpf1 protein.

### Claim 8 (depends on 7)

The nucleic acid/protein composition of claim 7, wherein the CRISPR Type V Cpf1 protein, the CRISPR Type II Cas9 protein, or the CRISPR Type V Cpf1 protein and CRISPR Type II Cas9 protein are catalytically inactive.

### Claim 9 (depends on 8)

The nucleic acid/protein composition of claim 8, further comprising one or more donor polynucleotides.