# US 12,180,487 B2 · Minicircle producing bacteria engineered to differentially methylate nucleic acid molecules therein

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- Number: US 12,180,487 B2
- Document ID: US12180487B2
- Status: Granted
- Kind: B2
- Application number: 17/428,585
- Filed: 2020-02-06
- Published: 2022-03-31
- Granted: 2024-12-31
- Earliest claimed: 2019-02-06
- Assignees: Fred Hutchinson Cancer Center
- Applicants: Fred Hutchinson Cancer Center
- Inventors: Christopher Johnston; Sean Cotton
- Web page: https://patentgrep.com/patent/US12180487B2

## CPC

- C12N 15/10: Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34)
- C12N 15/70: Vectors or expression systems specially adapted for E. coli
- C12N 1/205: Bacterial isolates
- C12N 9/1007: {Methyltransferases (general) (2.1.1.)}
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N 15/74: Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12R 2001/19: Escherichia coli
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12R: INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS

## Abstract

Embodiments include engineered minicircle-producing bacterium with differential methylation capability, as well as kits and compositions comprising the bacterium. Further described are methods of using of the bacterium for producing differentially methylated minicircle DNA, and for improving transformation efficiency of exogenous DNA in intractable bacteria.

## Claims

### Claim 1

An engineered, minicircle producing bacterium comprising:
a parental plasmid comprising a minicircle nucleic acid sequence comprising an exogenous nucleic acid sequence and a plurality of restriction sites outside of the minicircle nucleic acid sequence,
wherein at least one endogenous methyltransferase comprising at least one of a Dam methyltransferase, a Dem methyltransferase, a HsdM methyltransferase, or a combination thereof, is absent or non-functional in the engineered bacterium, such that the engineered bacterium has reduced DNA-methylation capability and the exogenous nucleic acid sequence lacks methylation at a plurality of methylation cites that would be methylated in a reference bacterium of the same species as the engineered bacterium.

### Claim 2 (depends on 1)

The engineered bacterium of claim 1, wherein the at least one endogenous methyltransferase is non-functional in the engineered bacterium, and the engineered bacterium comprises a modification in a gene encoding a respective endogenous methyltransferase of the at least one endogenous methyltransferase.

### Claim 3 (depends on 1)

The engineered bacterium of claim 1, wherein the at least one endogenous methyltransferase methylates a cytosine residue of a sequence CCWGG, wherein the W is A or T, the at least one endogenous methyltransferase methylates an adenosine residue of a sequence GATC, a sequence AACN6GTGC, or both, or the at least one endogenous methyltransferase methylates a cytosine residue and an adenosine residue.

### Claim 4 (depends on 1)

The engineered bacterium claim 1, wherein the engineered bacterium is Escherichia coli.

### Claim 5 (depends on 1)

The engineered bacterium of claim 1, further comprising at least one of an inducible ϕC31 integrase or an inducible I-SceI homing endonuclease.

### Claim 6 (depends on 1)

A kit comprising an engineered bacterium of claim 1.

### Claim 7 (depends on 1)

A method, comprising:
producing a minicircle comprising the exogenous DNA sequence comprised in the parental plasmid in a first bacterium that is an engineered bacterium of claim 1; and
transforming the minicircle into a second bacterium, the minicircle lacking methylation at a plurality of methylation sites that would be methylated in a reference bacterium of the same species as the engineered bacterium and, thereby, resisting degradation when transformed into the second bacterium.

### Claim 8

A method, comprising:
transforming a parental plasmid into an engineered bacterium,
wherein the parental plasmid comprises a minicircle nucleic acid sequence comprising an exogenous nucleic acid sequence and a plurality of restriction sites outside of the minicircle nucleic acid sequence, and
wherein at least one endogenous methyltransferase comprising at least one of a Dam methyltransferase, a Dem methyltransferase, a HsdM methyltransferase, or a combination thereof, is absent or non-functional in the engineered bacterium, such that the engineered bacterium has reduced DNA-methylation capability and the exogenous nucleic acid sequence lacks methylation at a plurality of methylation cites that would be methylated in a reference bacterium of the same species as the engineered bacterium; and
producing a minicircle comprising the minicircle nucleic acid sequence, wherein the minicircle lacks methylation at a plurality of methylation sites that would be methylated in a reference bacterium of the same species as the engineered bacterium.

### Claim 9 (depends on 8)

The method of claim 8, wherein at least one endogenous methyltransferase is non-functional in the engineered bacterium, and the engineered bacterium comprises a modification in a gene encoding a respective endogenous methyltransferase of the at least one endogenous methyltransferase.

### Claim 10 (depends on 8)

The method of claim 8, wherein the at least one endogenous methyltransferase methylates a cytosine residue of a sequence CCWGG, wherein the W is A or T, the at least one endogenous methyltransferase methylates an adenosine residue of a sequence GATC, a sequence AACN6GTGC, or both, or the at least one endogenous methyltransferase methylates a cytosine residue and an adenosine residue.

### Claim 11 (depends on 7)

The method of claim 7, wherein at least one endogenous methyltransferase is non-functional in the engineered bacterium, and the engineered bacterium comprises a modification in a gene encoding a respective endogenous methyltransferase of the at least one endogenous methyltransferase.

### Claim 12 (depends on 7)

The method of claim 7, wherein the at least one endogenous methyltransferase methylates a cytosine residue of a sequence CCWGG, wherein the W is A or T, the at least one endogenous methyltransferase methylates an adenosine residue of a sequence GATC, a sequence AACN6GTGC, or both, or the at least one endogenous methyltransferase methylates a cytosine residue and an adenosine residue.