# US 12,178,884 B2 · Recombinogenic nucleic acid strands in situ

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- Number: US 12,178,884 B2
- Document ID: US12178884B2
- Status: Granted
- Kind: B2
- Application number: 16/993,009
- Filed: 2020-08-13
- Published: 2021-03-04
- Granted: 2024-12-31
- Earliest claimed: 2016-01-10
- Assignees: SNIPR Technologies Limited
- Applicants: SNIPR Technologies Limited
- Inventors: Jasper Clube
- Web page: https://patentgrep.com/patent/US12178884B2

## CPC

- A61K 48/0066: {Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid}
- A61K 35/74: Bacteria (therapeutic use of a bacterial protein A61K38/00)
- A61P 31/04: Antibacterial agents
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/222: {Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes}
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N 15/905: {in yeast}
- C12N 15/907: {in mammalian cells}
- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- C12N 15/70: Vectors or expression systems specially adapted for E. coli
- C12N 15/74: Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N 15/902: {using homologous recombination}
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2800/80: Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}

## Abstract

The invention relates to retrieving or modifying target nucleic acids, such as host cell chromosomal DNA, by homologous recombination with vectors that have been cut to provide recombinogenic nucleic acid strands in situ. The methods described herein can be used to modify a target nucleic acid in vitro or in any prokaryotic or eukaryotic cell using homologous recombination.

## Claims

### Claim 1

A method for modifying a target nucleic acid strand in vitro, comprising:
(a) providing a nucleic acid vector comprising a first nucleic acid strand, wherein the first strand comprises:
i. a first homology arm (HA1) that is homologous to a first nucleotide sequence (NS1) of a donor strand for homologous recombination between the first strand and the donor strand;
ii. a second homology arm (HA2) that is homologous to a second nucleotide sequence (NS2) of the donor strand for homologous recombination between the first strand and the donor strand; and
iii. an intervening nucleotide sequence joining the 3′ end of HA1 to the 5′ end of HA2;
wherein HA1 is 5′ to HA2 in the first strand; and NS1 is 3′ to NS2 in the donor strand,
wherein the donor strand comprises a retrieval sequence (RS) between NS1 and NS2;
(b) combining the first strand with the donor strand;
(c) performing Cas nuclease cutting of a first cut site (CS1) of the first strand at the 5′ end of HA1, or flanking 5′ of the 5′ end of HA1; and/or Cas nuclease cutting of a second cut site (CS2) of the first strand at the 3′ end of HA2, or flanking 3′ of the 3′ end of HA2 to provide a cut first strand; and
wherein steps (b) and (c) are carried out in any order or simultaneously;
(d) carrying out homologous recombination of the cut first strand with the donor strand, whereby gap repair of the cut first strand produces a product vector in which RS is retrieved between HA1 and HA2, and wherein the product of step (d) is a circular vector; and
(e) inserting all or part of said RS of the product vector into the target nucleic acid strand;
wherein a mutation of the target nucleic acid strand is removed by said insertion of RS into the target nucleic acid strand.

### Claim 2 (depends on 1)

The method of claim 1, wherein the nucleic acid vector is a BAC, YAC, PAC, cosmid or plasmid.

### Claim 3 (depends on 1)

The method of claim 1, wherein the method uses a respective cutting site motif to direct each said cutting of step (c), wherein each cutting site is removed by the homologous recombination.

### Claim 4 (depends on 1)

The method of claim 1, wherein the donor strand is a strand of a chromosome or an episome of a first cell.

### Claim 5 (depends on 1)

The method of claim 1, wherein the RS is at least 10 kb in length.

### Claim 6 (depends on 1)

The method of claim 1, wherein in step (a) the vector comprises in 5′ to 3′ direction:
A. A first protospacer sequence (PS1) comprising CS1;
B. A first PAM (P1) adjacent to the 3′ end of PS1; and
C. HA1;
wherein a seed sequence (S1) of PS1 joins CS1 to P1; and/or
wherein in step (a) the vector comprises in 5′ to 3′ direction:
D. HA2;
E. A second protospacer sequence (PS2) comprising CS2;
F. A second PAM (P2) adjacent to the 3′ end of PS2;
wherein a seed sequence (S2) of PS2 joins CS2 to P2.

### Claim 7 (depends on 6)

The method of claim 6, wherein a protospacer/PAM combination at the 5′ end of HA1 or flanking 5′ of the 5′ end of HA1 is destroyed by step (d), whereby PS1 is rendered non-functional in the first strand; and/or wherein a protospacer/PAM combination at the 3′ end of HA2 or flanking 3′ of the 3′ end of HA2 is destroyed by step (d), whereby PS2 is rendered non-functional in the first strand.

### Claim 8 (depends on 1)

The method of claim 1, wherein in step (c) a guide RNA is used to guide the Cas nuclease cutting CS1, and the same guide RNA sequence is used to guide the Cas nuclease cutting of CS2.

### Claim 9 (depends on 1)

The method of claim 1, wherein before or during step (d) the donor strand is cut at the 5′ end of NS2 or flanking 5′ of the 5′ end of NS2; and/or at the 3′ end of NS1 or flanking 3′ of the 3′ end of NS1.

### Claim 10 (depends on 1)

The method of claim 1, wherein the target nucleic acid strand is comprised by a cell and the product vector of step (d) is introduced into the cell before insertion of the RS of the product vector into the target nucleic acid strand via homologous recombination.

### Claim 11 (depends on 10)

The method of claim 10, wherein the cell is a eukaryotic cell.

### Claim 12 (depends on 1)

The method of claim 1, wherein the nucleic acid vector provided in step (a) is a closed circular vector.

### Claim 13 (depends on 12)

The method of claim 12, wherein in step (b) the nucleic acid vector is supercoiled.

### Claim 14 (depends on 1)

The method of claim 1, wherein a sequence between HA1 and HA2 is deleted from the first strand in step (c) and/or a sequence between NS1 and NS2 is deleted from the donor strand in or after step (d).

### Claim 15 (depends on 1)

The method of claim 1, wherein the inserted RS sequence is functional for expression from the modified first strand.