# US 12,180,488 B2 · Recombination systems for high-throughput chromosomal engineering of bacteria

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- Number: US 12,180,488 B2
- Document ID: US12180488B2
- Status: Granted
- Kind: B2
- Application number: 16/960,147
- Filed: 2019-01-11
- Published: 2021-04-29
- Granted: 2024-12-31
- Earliest claimed: 2018-01-12
- Assignees: University of Massachusetts
- Applicants: University of Massachusetts
- Inventors: Kenan C. Murphy; Christopher Sassetti
- Web page: https://patentgrep.com/patent/US12180488B2

## CPC

- C12N 15/74: Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

In some aspects, the disclosure relates methods and compositions for genetic engineering of cells (e.g., bacterial cells, etc.). The disclosure is based, in part, on a combination of genetic recombination systems (e.g., a “targeting oligonucleotide” and a “payload plasmid) that enable high-throughput chromosomal engineering and do not involve the preparation of double-stranded DNA (dsDNA) recombination substrates. In some aspects, the disclosure provides engineered bacterial cells comprising a targeting oligonucleotide and a payload plasmid.

## Claims

### Claim 1

A method for genetic modification of a bacterium, the method comprising contacting a bacterium with:
(i) a single-stranded targeting oligonucleotide comprising an attP attachment site flanked by homology arms that are homologous to a target nucleic acid sequence of the bacterium; and,
(ii) a non-replicating payload plasmid comprising an attB attachment site and a nucleic acid sequence encoding a selectable marker,
wherein a single strand annealing protein (SSAP) and/or an integrase protein is expressed in the bacterium.

### Claim 2 (depends on 1)

The method of claim 1, wherein the bacterium is a Mycobacterium.

### Claim 3 (depends on 1)

The method of claim 1, wherein each homology arm comprises a nucleic acid sequence that is between 25 and 100 bases long.

### Claim 4 (depends on 1)

The method of claim 1, wherein the single-stranded targeting oligonucleotide comprises a sequence selected from SEQ ID NOs: 12 to 78.

### Claim 5 (depends on 1)

The method of claim 1, wherein the attP attachment site is a Bxb1 attP attachment site.

### Claim 6 (depends on 1)

The method of claim 1, wherein the attB attachment site is a Bxb1 attB attachment site.

### Claim 7 (depends on 1)

The method of claim 1, wherein the selectable marker comprises a nucleic acid sequence encoding a drug resistance gene.

### Claim 8 (depends on 1)

The method of claim 1, wherein the non-replicating payload plasmid further comprises a nucleic acid sequence encoding a promoter sequence or a peptide.

### Claim 9 (depends on 1)

The method of claim 1, wherein the integrase protein expressed in the bacterium is Bxb1 phage integrase.

### Claim 10 (depends on 1)

The method of claim 1, wherein the SSAP expressed in the bacterium is a RecT protein.

### Claim 11 (depends on 1)

The method of claim 1, wherein the bacterium is contacted simultaneously with the single-stranded targeting oligonucleotide and the non-replicating payload plasmid.

### Claim 12 (depends on 8)

The method of claim 8, wherein the contacting results in a deletion of the target nucleic acid sequence or an insertion of the nucleic acid sequence encoding the promoter sequence or the peptide into the genome of the bacterium.

### Claim 13

A bacterial cell comprising:
(i) a single-stranded targeting oligonucleotide comprising an attP attachment site flanked by homology arms that are homologous to a target nucleic acid sequence of the bacterial cell; and,
(ii) a non-replicating payload plasmid comprising an attB attachment site and a nucleic acid sequence encoding a selectable marker,
wherein a single strand annealing protein (SSAP) and/or an integrase protein is expressed in the bacterial cell.

### Claim 14 (depends on 13)

The bacterial cell of claim 13, wherein the bacterial cell is a Mycobacterium cell.

### Claim 15 (depends on 13)

The bacterial cell of claim 13, wherein the cell comprises a plasmid encoding the SSAP and/or the integrase protein.

### Claim 16 (depends on 13)

The bacterial cell of claim 13, wherein the targeting oligonucleotide comprises a sequence selected from SEQ ID NOs: 12 to 78.

### Claim 17 (depends on 13)

The bacterial cell of claim 13, wherein the non-replicating payload plasmid further comprises a nucleic acid sequence encoding a promoter sequence or a peptide.

### Claim 18 (depends on 13)

The bacterial cell of claim 13, wherein the integrase protein expressed in the bacterial cell is Bxb1 phage integrase.

### Claim 19 (depends on 13)

The bacterial cell of claim 13, wherein the SSAP expressed in the bacterial cell is a RecT protein.