Method for bacterial genome editing

Patent metadata

Field	Value
Publication number	US-2017114367-A1
Application number	US-201615066063-A
Country	US
Kind code	A1
Filing date	Mar 10, 2016
Priority date	Oct 26, 2015
Publication date	Apr 27, 2017
Grant date	—

Abstract

A method for bacterial genome editing includes: providing a bacterial cell; transforming a pCas9 plasmid and a pKD46 plasmid into the bacterial cell; co-transforming a pCRISPR::LacZ plasmid and an exogenous DNA into the bacterial cell carrying the pCas9 plasmid and the pKD46 plasmid, so as to obtain a strain broth; and spreading the strain broth on a culture medium to conduct cultivation.

First claim

What is claimed is:

1 . A method for bacterial genome editing, comprising following steps:
providing a bacterial cell; transforming a pCas9 plasmid and a pKD46 plasmid into the bacterial cell; co-transforming a pCRISPR::LacZ plasmid and an exogenous DNA into the bacterial cell carrying the pCas9 plasmid and the pKD46 plasmid, so as to obtain a strain broth; and spreading the strain broth on a culture medium to conduct cultivation.

2 . The method for bacterial genome editing as claimed in claim 1 , wherein after the steps of co-transforming the pCRISPR::LacZ plasmid and the exogenous DNA into the bacterial cell carrying the pCas9 plasmid and the pKD46 plasmid, further comprises following steps:
using a Cas9 protein and a guide RNA which can identify a LacZ gene to specifically cut at a specific cutting site of the LacZ gene in the bacterial cell; and inserting the exogenous DNA into the specific cutting site of the LacZ gene in the bacterial cell, and obtaining the strain broth.

3 . The method for bacterial genome editing as claimed in claim 1 , wherein the bacterial cell is an Escherichia coli.

4 . The method for bacterial genome editing as claimed in claim 3 , wherein the Escherichia coli is a bacterial strain of MG1655.

5 . The method for bacterial genome editing as claimed in claim 1 , wherein the guide RNA is expressed from the pCRISPR::LacZ plasmid which can identify the LacZ gene.

6 . The method for bacterial genome editing as claimed in claim 1 , wherein a method of transforming the pCas9 plasmid and the pKD46 plasmid into the bacterial cell is an electroporation method.

7 . The method for bacterial genome editing as claimed in claim 1 , wherein a method of co-transforming the pCRISPR::LacZ plasmid and the exogenous DNA into the bacterial cell carrying the pCas9 plasmid and the pKD46 plasmid is an electroporation method.

8 . The method for bacterial genome editing as claimed in claim 1 , wherein the culture medium contains isopropyl β-D-1-thiogalactopyranoside, 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, Kanamycin, and Tetracycline.

9 . The method for bacterial genome editing as claimed in claim 1 , wherein a cultivation condition of the strain broth is at 37° C. for 16 to 24 hours.

10 . A method for bacterial genome editing, comprising following steps:
providing a bacterial cell; co-transforming a pHR_trc template plasmid and a pCas9-NSI plasmid into the bacterial cell to obtain a strain broth; and spreading the strain broth on a culture medium to conduct cultivation.

11 . The method for bacterial genome editing as claimed in claim 10 , wherein the bacterial cell is a cyanobacteria.

12 . The method for bacterial genome editing as claimed in claim 11 , wherein the cyanobacteria is a bacterial strain of S. elongatus PCC7942.

13 . The method for bacterial genome editing as claimed in claim 10 , wherein the pHR_trc template plasmid is used as a homologous recombination template.

14 . The method for bacterial genome editing as claimed in claim 10 , wherein the pHR_trc template plasmid contains a Spectinomycin resistance gene Spec R , a fluorescent protein EGFP, and a homologous recombination region NSIa and NSIb.

15 . The method for bacterial genome editing as claimed in claim 10 , wherein the pCas9-NSI plasmid is used for correctly binding to a predetermined position to conduct a genetic recombination process.

16 . The method for bacterial genome editing as claimed in claim 15 , wherein the predetermined position is a double-strand break locus.

17 . The method for bacterial genome editing as claimed in claim 10 , wherein a method of co-transforming the pHR_trc template plasmid and the pCas9-NSI plasmid into the bacterial cell is phagocytosis.

18 . The method for bacterial genome editing as claimed in claim 10 , wherein the culture medium is a BG-11 culture medium containing Spectinomycin.

Assignees

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

Inventors

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

Classifications

• C12N15/72

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

• C12N15/74

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

• C12N15/902 Primary

  using homologous recombination · CPC title

• C12N2800/101

  for bacteria · CPC title

• C12N15/70 Primary

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