# US 10,711,267 B2 · Recombinant type I CRISPR-Cas system

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- Number: US 10,711,267 B2
- Document ID: US10711267B2
- Status: Granted
- Kind: B2
- Application number: 16/582,269
- Filed: 2019-09-25
- Published: 2020-04-02
- Granted: 2020-07-14
- Earliest claimed: 2018-10-01
- Assignees: North Carolina State University
- Applicants: North Carolina State University
- Inventors: Rodolphe Barrangou; Claudio Hidalgo-Cantabrana
- Web page: https://patentgrep.com/patent/US10711267B2

## CPC

- C12N 15/102: {Mutagenizing nucleic acids}
- C40B 40/06: Libraries containing nucleotides or polynucleotides, or derivatives thereof
- C12N 1/04: Preserving or maintaining viable microorganisms (immobilised microorganisms C12N11/00)
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/224: {Class 1 CAS enzyme complex, e.g. multi-enzyme}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 15/03: Bacteria
- 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 15/746: {for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)}
- C12N 15/90: Stable introduction of foreign DNA into chromosome
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2795/00061: Methods of inactivation or attenuation
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60)

## Abstract

This invention relates to recombinant Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) arrays and recombinant nucleic acid constructs encoding Type I-E CASCADE complexes as well as plasmids, retroviruses and bacteriophage comprising the same.

## Claims

### Claim 1

A recombinant nucleic acid construct comprising a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) array comprising two or more repeat sequences and one or more spacer sequence(s), wherein each of the one or more spacer sequences is linked at its 5′ end and at its 3′ end to a repeat sequence, and each of the one or more spacer sequences is complementary to one or more target sequences in a target DNA of a target organism, wherein the target DNA is located immediately adjacent (3′) to a protospacer adjacent motif (PAM), wherein the two or more repeat sequences comprise 20 to 28 consecutive nucleotides of any one of the nucleotide sequences of SEQ ID NOs:1, 10, 19, 28, 37, 42, 51, or 60.

### Claim 2 (depends on 1)

The recombinant nucleic acid construct of claim 1, wherein the PAM comprises a nucleotide sequence of 5′-NAA-,3′ 5′-AAA-3′ or 5′-AA-3′ that is immediately adjacent to and 5′ of the target sequence.

### Claim 3 (depends on 1)

The recombinant nucleic acid construct of claim 1, wherein the spacer sequence is about 80 to 100% complementary to the target sequence.

### Claim 4 (depends on 1)

The recombinant nucleic acid construct of claim 1, wherein the one or more spacer sequence(s) each have a length of about 25 nucleotides to about 40 nucleotides.

### Claim 5 (depends on 1)

The recombinant nucleic acid construct of claim 1, wherein the one or more spacer sequence(s) each comprise a 5′ region and a 3′ region, wherein the 5′ region comprises a seed sequence and the 3′ region comprises a remaining portion of the one or more spacer sequence(s).

### Claim 6 (depends on 5)

The recombinant nucleic acid construct of claim 5, wherein the seed sequence comprises the first 8 nucleotides of the 5′ end of each of the one or more spacer sequence(s), and is fully complementary to the target sequence, and the remaining portion of the one or more spacer sequence(s) is at least 80% complementary to the target sequence.

### Claim 7 (depends on 1)

The recombinant nucleic acid construct of claim 1, wherein the target sequence is located in a gene, optionally in the sense or coding strand or in the antisense or non-coding strand.

### Claim 8 (depends on 7)

The recombinant nucleic acid construct of claim 7, wherein the target sequence is located in an intragenic region of the gene, optionally located in the sense or coding strand or in the antisense or non-coding strand.

### Claim 9 (depends on 1)

The recombinant nucleic acid construct of claim 1, wherein the target sequence is located in an intergenic region.

### Claim 10 (depends on 7)

The recombinant nucleic acid construct of claim 7, wherein the target sequence is located on a chromosome, a mobile element, an extrachromosomal nucleic acid or a plasmid.

### Claim 11 (depends on 7)

The recombinant nucleic acid construct of claim 7, wherein the gene encodes a transcription factor or a promoter.

### Claim 12 (depends on 7)

The recombinant nucleic acid construct of claim 7, wherein the gene encodes non-coding RNA (e.g., miRNA, siRNA, piwi-interacting RNA (piRNA) and long non-coding RNA (lncRNA).

### Claim 13 (depends on 1)

The recombinant nucleic acid construct of claim 1, wherein the target organism is a prokaryote or a eukaryote.

### Claim 14 (depends on 1)

A recombinant cell comprising the recombinant nucleic acid construct of claim 1.

### Claim 15 (depends on 1)

A vector encoding the recombinant nucleic acid of claim 1.

### Claim 16 (depends on 15)

The vector of claim 15, further comprising a recombinant nucleic acid encoding a Type I-E CRISPR associated complex for antiviral defense complex (Cascade complex) comprising a Cse1 polypeptide, a Cse2 polypeptide, a Cas7 polypeptide, a Cas5 polypeptide and a Cas6 polypeptide.

### Claim 17 (depends on 16)

The vector of claim 16, wherein the Cse1 polypeptide is encoded by the nucleotide sequence of SEQ ID NO:82, the Cse2 polypeptide is encoded by the nucleotide sequence of SEQ ID NO:83, the Cas7 polypeptide is encoded by the nucleotide sequence of SEQ ID NO:84, the Cas5 polypeptide is encoded by the nucleotide sequence of SEQ ID NO:85, and the Cas6 polypeptide is encoded by the nucleotide sequence of SEQ 11) NO:86.

### Claim 18 (depends on 16)

The vector of claim 16, further comprising a polynucleotide encoding a Cas3 polypeptide, wherein the polynucleotide encoding the Cas3 comprises the nucleotide sequence of SEQ ID NO:87 or wherein the polynucleotide encoding the Cas3 encodes the amino acid sequence of SEQ ID NO:119.

### Claim 19 (depends on 15)

The vector of claim 15, wherein the vector is a plasmid, bacteriophage, and/or retrovirus.