# US 11,441,149 B2 · CRISPR/cas systems for c1-fixing bacteria

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- Number: US 11,441,149 B2
- Document ID: US11441149B2
- Status: Granted
- Kind: B2
- Application number: 15/442,695
- Filed: 2017-02-26
- Published: 2017-08-31
- Granted: 2022-09-13
- Earliest claimed: 2016-02-26
- Assignees: LanzaTech NZ, Inc.
- Applicants: LanzaTech NZ, Inc.
- Inventors: Shilpa Nagaraju; Michael Koepke
- Web page: https://patentgrep.com/patent/US11441149B2

## CPC

- C12N 15/74: Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N 15/102: {Mutagenizing nucleic acids}
- C12N 15/635: {Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline}
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- Y02E 50/10: Biofuels, e.g. bio-diesel
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION

## Abstract

The invention provides methods of genetically engineering a C1-fixing bacterium using a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) (CRISPR/Cas) system. Preferably, the Cas protein is under the control of an inducible promoter.

## Claims

### Claim 1

A method of genetically engineering a C1-fixing bacterium comprising introducing into a C1-fixing bacterium containing a DNA molecule comprising a target sequence an engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) (CRISPR/Cas) system comprising one or more vectors comprising:
(a) a nucleotide sequence encoding a guide RNA that hybridizes with the target sequence and
(b) a nucleotide sequence encoding a type-II Cas9 protein under the control of an inducible promoter;
wherein the inducible promoter is ipl12;
wherein the Cas9 interacts with the DNA molecule; and
wherein expression of a gene product or a nucleotide sequence of the DNA molecule is altered.

### Claim 2 (depends on 1)

The method of claim 1, wherein the CRISPR/Cas system further comprises on the one or more vectors:
(c) a nucleotide sequence comprising a 5′ homology arm that hybridizes upstream of the target sequence and a 3′ homology arm that hybridizes downstream of the target sequence,
whereby the 5′ homology arm and the 3′ homology arm hybridize with the DNA molecule and homologous recombination occurs, resulting in the replacement of the target sequence with DNA located between the 5′ homology arm and the 3′ homology arm.

### Claim 3 (depends on 2)

The method of claim 2, wherein the DNA located between the 5′ homology arm and the 3′ homology arm disrupts the DNA molecule, whereby expression of the gene product of the DNA molecule is decreased.

### Claim 4 (depends on 2)

The method of claim 2, wherein the DNA located between the 5′ homology arm and the 3′ homology arm encodes an exogenous gene, whereby the homologous recombination inserts the exogenous gene into the DNA molecule.

### Claim 5 (depends on 4)

The method of claim 4, wherein the C1-fixing bacterium expresses the exogenous gene.

### Claim 6 (depends on 2)

The method of claim 2, wherein (a), (b), and (c) are located on the same or different vectors.

### Claim 7 (depends on 1)

The method of claim 1, wherein the Cas9 is catalytically active.

### Claim 8 (depends on 1)

The method of claim 1, wherein the Cas9 is nickase Cas9.

### Claim 9 (depends on 1)

The method of claim 1, wherein the Cas9 is catalytically inactive.

### Claim 10 (depends on 1)

The method of claim 1, wherein the Cas9 cleaves the DNA molecule and expression of the gene product of the DNA molecule is decreased.

### Claim 11 (depends on 1)

The method of claim 1, wherein the Cas9 blocks the DNA molecule and expression of the gene product of the DNA molecule is decreased.

### Claim 12 (depends on 1)

The method of claim 1, wherein (a) and (b) are located on the same or different vectors.

### Claim 13 (depends on 1)

The method of claim 1, wherein the CRISPR/Cas system is derived from Streptococcus pyogenes or Streptococcus thermophilus.

### Claim 14 (depends on 1)

The method of claim 1, wherein the C1-fixing bacterium is selected from the group consisting of Acetobacterium woodii, Alkalibaculum bacchii, Blautia producta, Butyribacterium methylotrophicum, Clostridium aceticum, Clostridium autoethanogenum, Clostridium carboxidivorans, Clostridium coskatii, Clostridium drakei, Clostridium formicoaceticum, Clostridium ljungdahlii, Clostridium magnum, Clostridium ragsdalei, Clostridium scatologenes, Eubacterium limosum, Moorella thermautotrophica, Moorella thermoacetica, Oxobacter pfennigii, Sporomusa ovata, Sporomusa silvacetica, Sporomusa sphaeroides, and Thermoanaerobacter kiuvi.

### Claim 15 (depends on 1)

The method of claim 1, wherein the C1-fixing bacterium is Clostridium autoethanogenum, Clostridium ljungdahlii, or Clostridium ragsdalei.

### Claim 16 (depends on 1)

The method of claim 1, wherein the C1-fixing bacterium is Clostridium autoethanogenum.