Minicircle producing bacteria engineered to differentially methylate nucleic acid molecules therein

Patent metadata

Field	Value
Publication number	US-12180487-B2
Application number	US-202017428585-A
Country	US
Kind code	B2
Filing date	Feb 6, 2020
Priority date	Feb 6, 2019
Publication date	Dec 31, 2024
Grant date	Dec 31, 2024

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.

First claim

The invention claimed is:

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.

2. 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.

3. 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 AACN 6 GTGC, or both, or the at least one endogenous methyltransferase methylates a cytosine residue and an adenosine residue.

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

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

6. A kit comprising an engineered bacterium of claim 1 .

7. 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.

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.

9. 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.

10. 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.

11. 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.

12. 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.

Assignees

• Fred Hutchinson Cancer Center

Inventors

• Johnston Christopher
• Cotton Sean

Classifications

• C12N15/74

  Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora · CPC title

• C12N9/1007

  Methyltransferases (general) (2.1.1.) · CPC title

• C12R2001/19

  Escherichia coli · CPC title

• C12N1/205

  Bacterial isolates · CPC title

• C12N15/70 Primary

  Vectors or expression systems specially adapted for E. coli · CPC title