Cas9 plasmid, genome editing system and method of Escherichia coli

Patent metadata

Field	Value
Publication number	US-9988637-B2
Application number	US-201715495580-A
Country	US
Kind code	B2
Filing date	Apr 24, 2017
Priority date	Oct 26, 2015
Publication date	Jun 5, 2018
Grant date	Jun 5, 2018

Abstract

A Cas9 expression plasmid, a genome editing system and a genome editing method for Escherichia coli are provided. The Cas9 expression plasmid includes a tracrRNA sequence, a Cas9 gene and a chloramphenicol resistance gene (Cm R ). The Cas9 expression plasmid is applied to CRISPR/Cas-coupled λ-red recombineering system for editing genomes of E. coli with high efficiency.

First claim

What is claimed is:

1. A Cas9 expression plasmid, comprising a nucleotide sequence of SEQ ID NO: 1 including a tracrRNA sequence, a Cas9 gene sequence and a chloramphenicol resistance gene (Cm R ) sequence.

2. A genome editing system for Escherichia coli ( E. coli ), comprising:
an E. coli strain;
a Cas9 expression plasmid according to claim 1 ;
a λ-red recombinase expression plasmid including a P araB promoter, a Gam gene, a Bet gene and an Exo gene sequentially;
a crRNA expression plasmid including a promoter sequence, a crRNA sequence and a spacer sequence, wherein the spacer sequence is complementary to a first specific sequence on a chromosome of the E. coli strain; and
a linear DNA including a right homology arm (HRR), a donor DNA and a left homology arm (HRL), wherein the right homology arm (HRR) and the left homology arm (HRL) cooperatively form a homologous recombination region of which the sequence is complementary to a second specific sequence on the chromosome of the E. coli strain.

3. The genome editing system for E. coli according to claim 2 , wherein the crRNA expression plasmid further includes a tracrRNA sequence, and the tracrRNA sequence and the spacer sequence cooperatively form a single guide RNA (sgRNA) sequence.

4. The genome editing system for E. coli according to claim 2 , wherein the size of the right homology arm is the same as that of the left homology arm and is between 40 bp and 80 bp.

5. The genome editing system for E. coli according to claim 2 , wherein the linear DNA further includes a first antibiotic resistant gene.

6. The genome editing system for E. coli according to claim 5 , wherein the first antibiotic resistant gene is tetracycline resistance gene (Tc R ).

7. The genome editing system for E. coli according to claim 2 , wherein the E. coli strain is selected from K-12 strain and W strain.

8. The genome editing system for E. coli according to claim 7 , wherein the E. coli strain is selected from MG1655, WΔ5 and WΔ5#1 strains.

9. A genome editing method for Escherichia coli , comprising steps of:
providing an E. coli strain;
constructing a Cas9 expression plasmid having a nucleotide sequence of SEQ ID NO: 1, wherein the Cas9 expression plasmid includes a tracrRNA sequence, Cas9 gene sequence and a chloramphenicol resistance gene (Cm R ) sequence;
constructing a λ-red recombinase expression plasmid sequentially harboring a P araB promoter, a Gam gene, a Bet gene and an Exo gene;
constructing a crRNA expression plasmid harboring a promoter sequence, a crRNA sequence and a spacer sequence, wherein the spacer sequence is complementary to a first specific sequence on a chromosome of the E. coli strain;
preparing a linear DNA including a right homology arm (HRR), a donor DNA and a left homology arm (HRL), the right homology arm and the left homology arm cooperatively forming a homologous recombination region of which the sequence is complementary to a second specific sequence on the chromosome of the E. coli strain;
co-transforming the Cas9 expression plasmid and the λ-red recombinase expression plasmid into the E. coli strain to produce a first transformant;
triggering the expression of Gam, Exo, and Beta proteins of the λ-red recombinase expression plasmid by adding arabinose;
co-transforming the crRNA expression plasmid and the linear DNA into the first transformant to obtain a second transformant; and
incubating the second transformat, wherein the Cas9 expression plasmid expresses a tracrRNA and a Cas9 protein, the crRNA expression plasmid expresses a crRNA; the tracrRNA, the Cas9 protein and the crRNA cooperatively form a Cas9 protein complex to produce a double-stranded break specific to the first specific sequence of the second transformant; then, the homologous recombination region and the second specific sequence of the second transformant undergo homologous recombination to insert the donor DNA into the first specific sequence of the second transformant.

10. The genome editing method for E. coli according to claim 9 , wherein the crRNA expression plasmid further includes a tracrRNA sequence, and the tracrRNA sequence and the spacer sequence cooperatively form a single guide RNA (sgRNA) sequence.

11. The genome editing method for E. coli according to claim 9 , wherein the linear DNA further includes a first antibiotic resistant gene.

12. The genome editing method for E. coli according to claim 11 , further includes a recovery step, wherein the second transformant is cultured in an antibiotic-free medium for 2 to 3 hours.

13. The genome editing method for E. coli according to claim 12 , further includes a screening step, wherein the second transformant is cultured in a medium containing a first antibiotics after the recovery step.

14. The genome editing method for E. coli according to claim 13 , wherein the first antibiotics is tetracycline.

15. The genome editing method for E. coli according to claim 9 , wherein the step of preparing the linear DNA further includes:
providing a template plasmid;
performing PCR-amplification of the template plasmid by a primer pair to obtain a PCR product, wherein the primer pair is composed of a forward primer and a reverse primer, the 5′-end of the forward primer has a sequence of the left homology arm and the 5′-end of the reverse primer has a sequence that is complementary to the right homology arm;
purifying the PCR product to obtain a DNA solution; and
eluting the DNA solution to obtain the linear DNA.

16. The genome editing method for E. coli according to claim 15 , wherein the DNA solution is eluted through a membrane filter with a pore size of 0.025 μm.

Assignees

• Univ Nat Tsing Hua
• Chang Chun Plastics Co Ltd
• Chang Chun Petrochemical Co Ltd
• Nat Tsing Hua Univeristy

Inventors

• Hu yu chen
• Chung Mu-En
• Yeh I-Hsin
• Li Hung
• Sung Li-Yu

Classifications

• C12N15/102 Primary

  Mutagenizing nucleic acids · CPC title

• C12N15/72

  Expression systems using regulatory sequences derived from the lac-operon · CPC title

• C12N15/73 Primary

  Expression systems using phage (lambda) regulatory sequences · CPC title

• C12N9/22

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N2800/101

  for bacteria · CPC title