Optimization and Application of Double-plasmid CRISPR-Cas9 System in Escherichia coli

doi:10.13560/j.cnki.biotech.bull.1985.2021-0618

Abstract

Abstract:

In recent years,the CRISPR-Cas9（clustered regularly interspaced short palindromic repeats）system has been successfully exploited as versatile genome editing tools in various microorganisms. Because the application of CRISPR-Cas9 system is restricted only by the NGG of PAM（protospacer adjacent motif）sequence,therefore CRISPR-Cas9 system can theoretically edit any site or gene on the genome harboring NGG sequence. However,for the genes that have profound effects on cell growth and metabolism,the editing efficiency would be significantly reduced or even the mutants could not be obtained. Plenty of previous reports have provided valuable strategies to reduce the off-target effects of CRISPR-Cas9 system,but the reduction of editing efficiency is far from being solved. In this study,an efficient double- plasmid CRISPR-Cas9 system was established by using plasmids with different copy numbers to regulate the concentration of homology arms and the expression of Cas9 protein and gRNA,which would make them work more collaboratively for the gene editing purpose. The experimental results showed that the pfkA（6-phosphofructokinase isozyme 1）and pfkB（6-phosphofructokinase isozyme 2）genes in glycolysis pathway and the zwf（Glucose-6-phosphate 1-dehydrogenase）gene in pentose phosphate pathway were successfully deleted using the optimized double-plasmid CRISPR-Cas9 system,with the gene deletion efficiency of up to 100%;the nagABE gene cluster was also successfully replaced by glycerol kinase gene glpK,with the gene integration efficiency of 10%. By contrast,when using the single-plasmid CRISPR-Cas9 system,the pfkB gene deletion and glpK gene integration were successful in double-plasmid CRISPR-Cas9 system,and the efficiency of deleting pfkA and zwf increased by 31% and 63% respectively. The differences in carbon source utilization between mutant and wild-type strains further indicated that the gene editing efficiency was associated with special gene activity. Results from this study demonstrated that the sufficient target gene homology arms and the over-expression of gRNA may efficiently enhance the gene editing efficiency of CRISPR-Cas9 system.

Key words: CRISPR-Cas9 system, Escherichia coli, gene editing, double-plasmid system

WANG Kai-kai, WANG Xiao-lu, SU Xiao-yun, ZHANG Jie. Optimization and Application of Double-plasmid CRISPR-Cas9 System in Escherichia coli[J]. Biotechnology Bulletin, 2021, 37(12): 252-264.

Figures/Tables 10

References 33

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菌株Strain/质粒 Plasmid	功能 Function	来源 Source
菌株Strain
大肠杆菌Escherichia coli Top10	克隆宿主	天根
MG1655（DE3）	基本菌株	本研究
MG1655-∆pfkA	来源于MG1655（DE3）,pfkA缺失突变株	本研究
MG1655-∆pfkB	来源于MG1655（DE3）,pfkB缺失突变株	本研究
MG1655-∆zwf	来源于MG1655（DE3）,zwf缺失突变株	本研究
MG1655-∆ABE∷glpK	来源于MG1655（DE3）,nagABE基因位点替换为glpK的突变株	本研究
质粒 Plasmid
pRed_cas9_recA_poxb300	Cas9供体质粒	MolecularCloud
p∆pfkA	pfkA同源臂-gRNA供体质粒,用于双质粒CRISPR-Cas9系统	本研究
pRA∆pfkA	pfkA敲除质粒,用于单质粒CRISPR-Cas9系统	本研究
p∆pfkB	pfkB同源臂-gRNA供体质粒,用于双质粒CRISPR-Cas9系统	本研究
pRA∆pfkB	pfkB敲除质粒,用于单质粒CRISPR-Cas9系统	本研究
p∆zwf	zwf同源臂-gRNA供体质粒,用于双质粒CRISPR-Cas9系统	本研究
pRA∆zwf	zwf敲除质粒,用于单质粒CRISPR-Cas9系统	本研究
p∆ABE∷glpK	nagABE同源臂-gRNA供体质粒,用于双质粒CRISPR-Cas9系统	本研究
pRA∆ABE∷glpK	nagABE敲除质粒,用于单质粒CRISPR-Cas9系统	本研究

菌株Strain/质粒 Plasmid	功能 Function	来源 Source
菌株Strain
大肠杆菌Escherichia coli Top10	克隆宿主	天根
MG1655（DE3）	基本菌株	本研究
MG1655-∆pfkA	来源于MG1655（DE3）,pfkA缺失突变株	本研究
MG1655-∆pfkB	来源于MG1655（DE3）,pfkB缺失突变株	本研究
MG1655-∆zwf	来源于MG1655（DE3）,zwf缺失突变株	本研究
MG1655-∆ABE∷glpK	来源于MG1655（DE3）,nagABE基因位点替换为glpK的突变株	本研究
质粒 Plasmid
pRed_cas9_recA_poxb300	Cas9供体质粒	MolecularCloud
p∆pfkA	pfkA同源臂-gRNA供体质粒,用于双质粒CRISPR-Cas9系统	本研究
pRA∆pfkA	pfkA敲除质粒,用于单质粒CRISPR-Cas9系统	本研究
p∆pfkB	pfkB同源臂-gRNA供体质粒,用于双质粒CRISPR-Cas9系统	本研究
pRA∆pfkB	pfkB敲除质粒,用于单质粒CRISPR-Cas9系统	本研究
p∆zwf	zwf同源臂-gRNA供体质粒,用于双质粒CRISPR-Cas9系统	本研究
pRA∆zwf	zwf敲除质粒,用于单质粒CRISPR-Cas9系统	本研究
p∆ABE∷glpK	nagABE同源臂-gRNA供体质粒,用于双质粒CRISPR-Cas9系统	本研究
pRA∆ABE∷glpK	nagABE敲除质粒,用于单质粒CRISPR-Cas9系统	本研究

说明Instruction	引物Primer	寡核苷酸序列 Oligonucleotide sequence（5'-3'）
双质粒pfkA基因敲除pfkA gene knockout（double plasmid system）
gRNA扩增 gRNA amplification	pfkA-gRNA-F	TTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCGTGTCTGACAT-GATCAACCGGTTTTAGAGCTAGAAATAGCAAG
	gRNA-R	TCGGATCCACTAGTAACCATC
上游同源臂扩增Upstream homology arm amplification	pfkA-N-F	AATCACTAGTGAATTCGCGGCCGTCGGCATCTATATTTTATATAGCG
	pfkA-N-R	GAAATCAGACTACCTCTGAACTTTGGAATGCAAAATGAAATCTGTTGC
下游同源臂扩增Downstream homology arm amplification	pfkA-C-F	TCCAAAGTTCAGAGGTAGTCTGATTTCGGAAAAAGGCAGATTC
	pfkA-C-R	ATGCATCCAACGCGTTGGGAGCTCTCCCACCGTGTGACTGACGAATC
单质粒pfkA基因敲除pfkA gene knockout（single plasmid system）
	pfkA-F1	GATCTGTGTCTGACATGATCAACCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAG
	pfkA-R1	TATATAAAATATAGATGCCGAGGTCCCGCTTTGTTACAGAATGCTT
	pfkA-up-F1	CGGGACCTCGGCATCTATATTTTATATAGCGCGTTACGCATGG
	pfkA-down-R1	CGTTTTTGTCATGGCTTTCACCGTGTGACTGACG
	pfkA-F2	TGAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTAT
	pfkA-R2	CGGTTGATCATGTCAGACACAGATCTGACTCCATAACAGAGTACTCGC
双质粒pfkB基因敲除pfkB gene knockout（double plasmid system）
gRNA扩增 gRNA amplification	pfkB-gRNA-F	TTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCCACGTACATGTGGAAGCAAG-GTTTTAGAGCTAGAAATAGCAAG
	gRNA-R	TCGGATCCACTAGTAACCATC
上游同源臂扩增Upstream homology arm amplification	pfkB-N-F	CAATCACTAGTGAATTCGCGGCCGCAGCGACCAGGCAGTGGTGTGTC
	pfkB-N-R	GGGGAATGTTTTTGCATTTCCTCCTATAGGCTGA
下游同源臂扩增Downstream homology arm amplification	pfkB-C-F	GGAGGAAATGCAAAAACATTCCCCCAGCATTGGGGGAATCATCAC
	pfkB-C-R	TGCATCCAACGCGTTGGGAGCTCTCCTCAAAAGACGACTGATTGCCTGC
单质粒pfkB基因敲除pfkB gene knockout（single plasmid system）
	pfkB-F1	AGATCTCACGTACATGTGGAAGCAAGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAG
	pfkB-R1	ACACCACTGCCTGGTCGCTGGGTCCCGCTTTGTTACAGAATGCTTTTAAT
	pfkB-up-F1	AAAGCGGGACCCAGCGACCAGGCAGTGGTGTGTCCATACCAGGTCAT
	pfkB-down-R1	GCGTTTTTGTCATGGCTTTTCAAAAGACGACTGATTGCCTGCCACAT
	pfkB-F2	TTGAAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTATAATCA
	pfkB-R2	CTTGCTTCCACATGTACGTGAGATCTGACTCCATAACAGAGTACTCG
双质粒zwf基因敲除zwf gene knockout（double plasmid system）
gRNA扩增 gRNA amplification	zwf-gRNA-F	TTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCGCGTGCTGACTGGGATA- AAGGTTTTAGAGCTAGAAATAGCAAG
	gRNA-R	TCGGATCCACTAGTAACCATC
上游同源臂扩增Upstream homology arm amplification	zwf-N-F1	AATCACTAGTGAATTCGCGGCCGCGTGTCCATGCTGCGACAGAAACG
	zwf-N-R1	GCAGATAGTCATTCTCCTTAAGTTAACTAACCCGGTACTTAAGC
下游同源臂扩增Downstream homology arm amplification	zwf-C-F1	AGTTAACTTAAGGAGAATGACTATCTGCGCTTATCCTTTATGG
	zwf-C-R1	TCCAACGCGTTGGGAGCTCTCCCGCATTCGCTTCATGCAGGGCTTTAC
单质粒zwf基因敲除zwf gene knockout（single plasmid system）
	zwf-F1	ATGGAGTCAGATCTGCGTGCTGACTGGGATAAAGGTTTTAG- AGCTAGAAATAGCAAGTTAAAATAAGG
	zwf-R1	TCTGTCGCAGCATGGACACGGGTCCCGCTTTGTTACAGAATGCT
	zwf -up-F1	AAGCGGGACCCGTGTCCATGCTGCGACAGAAACGATTCACCGTCG
	zwf -down-R1	GTTTTTGTCATGGCTTTCGCATTCGCTTCATGCAGGGCTTTACGAAT
	zwf -F2	ATGAAGCGAATGCGAAAGCCATGACAAAAACGCGTAACAAAAGTG
	zwf -R2	CTTTATCCCAGTCAGCACGCAGATCTGACTCCATAACAGAGTACTCGCCTAT
双质粒glpK基因与nagABE替换nagABE replacement by glpK gene（double plasmid system）
gRNA扩增 gRNA amplification	nagABE-gRNA-F	TTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCCCTGCAGCGCCAG- TGCTTTCGTTTTAGAGCTAGAAATAGCAAG
	gRNA-R	TCGGATCCACTAGTAACCATC
上游同源臂扩增Upstream homology arm amplification	nagABE-N-F	AATCACTAGTGAATTCGCGGCCGAGTTTTCTACCTGAATATGCGGC
	nagABE-N-R	CTTATTGAGGTGAATAATGACACCAGGCGGACAAGCTCAGATAGG
下游同源臂扩增Downstream homology arm amplification	nagABE-C-F	TTGGTGACAAAACTCACAATCTGCTTTATGCCTGATGCGACGC
	nagABE-C-R	TGCATCCAACGCGTTGGGAGCTCTCCGGAGCTGATCAAAATAATCGG
nagB启动子扩增nagB promotor amplification	nagB-Pm-F	TGAGCTTGTCCGCCTGGTGTCATTATTCACCTCAATAAGTAAAATGTA
	nagB-Pm-R	CAAGCGTCGCATCAGGCATAAAGCAGATTGTGAGTTTTGTCACC
T7启动子扩增 T7 promotor amplification	nagABE-T7-F	TTTGGTGACAAAACTCACAAGGATCGAGATCTCGATCCCGCGAA
	nagABE-T7-R	GCAACGATATATTTTTTTTCAGTCATGGTATATCTCCTTCTTAAAGTT
glpK基因扩增 glpK gene amplification	pichi-glpk-F	CTTTAAGAAGGAGATATACCATGGGAAAAGACTATACACCAC
	pichi-glpk-R	CGTCGCATCAGGCATAAAGCAGATTAAGCAGTGTCCTTAAGCCAGCCC
单质粒glpK基因整合glpK gene integration（single plasmid system）
	ABE-F1	GTCAGATCTCCTGCAGCGCCAGTGCTTTCGTTTTAGAGCTAGAAATAGCAAGTTAAAA- TAAGG
	ABE -R1	GCATATTCAGGTAGAAAACTGGTCCCGCTTTGTTACAGAATGC
	ABE-up-F1	GCGGGACCAGTTTTCTACCTGAATATGCGGCATGTAATGAATTTTGCCGCTGTC
	ABE-down-R1	TGTTACGCGTTTTTGTCATGGCTTTGGAGCTGATCAAAATAATCGGAG
	ABE-F2	CTCCAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTATAATCACGGC
	ABE-R2	CGAAAGCACTGGCGCTGCAGGAGATCTGACTCCATAACAGAGTACTC
突变株检测引物Detection primer for mutants
	zwf-JC-F	ACGGCACAAACACCGCAGGC
	zwf-JC-R	ACATGATCAAGCGTTGCCATTGC
	A-JC-F	TTATCCTTGTCTCGTTTCAGCGTTGGGTGGTGCG
	A-JC-R	GTTTGATCCACTCTTTATCAATC
	B-JC-F	CATCGCGCTCTCGATAGCCGTTAT
	B-JC-R	GCGCAAATGCCATGCGGCATGGA
	ABE-JC-R	TTCAACGCTGCGGTCGCGGTAC
	ABE-JC-F	GGTTCCGCGATGGACACGCACC

说明Instruction	引物Primer	寡核苷酸序列 Oligonucleotide sequence（5'-3'）
双质粒pfkA基因敲除pfkA gene knockout（double plasmid system）
gRNA扩增 gRNA amplification	pfkA-gRNA-F	TTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCGTGTCTGACAT-GATCAACCGGTTTTAGAGCTAGAAATAGCAAG
	gRNA-R	TCGGATCCACTAGTAACCATC
上游同源臂扩增Upstream homology arm amplification	pfkA-N-F	AATCACTAGTGAATTCGCGGCCGTCGGCATCTATATTTTATATAGCG
	pfkA-N-R	GAAATCAGACTACCTCTGAACTTTGGAATGCAAAATGAAATCTGTTGC
下游同源臂扩增Downstream homology arm amplification	pfkA-C-F	TCCAAAGTTCAGAGGTAGTCTGATTTCGGAAAAAGGCAGATTC
	pfkA-C-R	ATGCATCCAACGCGTTGGGAGCTCTCCCACCGTGTGACTGACGAATC
单质粒pfkA基因敲除pfkA gene knockout（single plasmid system）
	pfkA-F1	GATCTGTGTCTGACATGATCAACCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAG
	pfkA-R1	TATATAAAATATAGATGCCGAGGTCCCGCTTTGTTACAGAATGCTT
	pfkA-up-F1	CGGGACCTCGGCATCTATATTTTATATAGCGCGTTACGCATGG
	pfkA-down-R1	CGTTTTTGTCATGGCTTTCACCGTGTGACTGACG
	pfkA-F2	TGAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTAT
	pfkA-R2	CGGTTGATCATGTCAGACACAGATCTGACTCCATAACAGAGTACTCGC
双质粒pfkB基因敲除pfkB gene knockout（double plasmid system）
gRNA扩增 gRNA amplification	pfkB-gRNA-F	TTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCCACGTACATGTGGAAGCAAG-GTTTTAGAGCTAGAAATAGCAAG
	gRNA-R	TCGGATCCACTAGTAACCATC
上游同源臂扩增Upstream homology arm amplification	pfkB-N-F	CAATCACTAGTGAATTCGCGGCCGCAGCGACCAGGCAGTGGTGTGTC
	pfkB-N-R	GGGGAATGTTTTTGCATTTCCTCCTATAGGCTGA
下游同源臂扩增Downstream homology arm amplification	pfkB-C-F	GGAGGAAATGCAAAAACATTCCCCCAGCATTGGGGGAATCATCAC
	pfkB-C-R	TGCATCCAACGCGTTGGGAGCTCTCCTCAAAAGACGACTGATTGCCTGC
单质粒pfkB基因敲除pfkB gene knockout（single plasmid system）
	pfkB-F1	AGATCTCACGTACATGTGGAAGCAAGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAG
	pfkB-R1	ACACCACTGCCTGGTCGCTGGGTCCCGCTTTGTTACAGAATGCTTTTAAT
	pfkB-up-F1	AAAGCGGGACCCAGCGACCAGGCAGTGGTGTGTCCATACCAGGTCAT
	pfkB-down-R1	GCGTTTTTGTCATGGCTTTTCAAAAGACGACTGATTGCCTGCCACAT
	pfkB-F2	TTGAAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTATAATCA
	pfkB-R2	CTTGCTTCCACATGTACGTGAGATCTGACTCCATAACAGAGTACTCG
双质粒zwf基因敲除zwf gene knockout（double plasmid system）
gRNA扩增 gRNA amplification	zwf-gRNA-F	TTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCGCGTGCTGACTGGGATA- AAGGTTTTAGAGCTAGAAATAGCAAG
	gRNA-R	TCGGATCCACTAGTAACCATC
上游同源臂扩增Upstream homology arm amplification	zwf-N-F1	AATCACTAGTGAATTCGCGGCCGCGTGTCCATGCTGCGACAGAAACG
	zwf-N-R1	GCAGATAGTCATTCTCCTTAAGTTAACTAACCCGGTACTTAAGC
下游同源臂扩增Downstream homology arm amplification	zwf-C-F1	AGTTAACTTAAGGAGAATGACTATCTGCGCTTATCCTTTATGG
	zwf-C-R1	TCCAACGCGTTGGGAGCTCTCCCGCATTCGCTTCATGCAGGGCTTTAC
单质粒zwf基因敲除zwf gene knockout（single plasmid system）
	zwf-F1	ATGGAGTCAGATCTGCGTGCTGACTGGGATAAAGGTTTTAG- AGCTAGAAATAGCAAGTTAAAATAAGG
	zwf-R1	TCTGTCGCAGCATGGACACGGGTCCCGCTTTGTTACAGAATGCT
	zwf -up-F1	AAGCGGGACCCGTGTCCATGCTGCGACAGAAACGATTCACCGTCG
	zwf -down-R1	GTTTTTGTCATGGCTTTCGCATTCGCTTCATGCAGGGCTTTACGAAT
	zwf -F2	ATGAAGCGAATGCGAAAGCCATGACAAAAACGCGTAACAAAAGTG
	zwf -R2	CTTTATCCCAGTCAGCACGCAGATCTGACTCCATAACAGAGTACTCGCCTAT
双质粒glpK基因与nagABE替换nagABE replacement by glpK gene（double plasmid system）
gRNA扩增 gRNA amplification	nagABE-gRNA-F	TTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCCCTGCAGCGCCAG- TGCTTTCGTTTTAGAGCTAGAAATAGCAAG
	gRNA-R	TCGGATCCACTAGTAACCATC
上游同源臂扩增Upstream homology arm amplification	nagABE-N-F	AATCACTAGTGAATTCGCGGCCGAGTTTTCTACCTGAATATGCGGC
	nagABE-N-R	CTTATTGAGGTGAATAATGACACCAGGCGGACAAGCTCAGATAGG
下游同源臂扩增Downstream homology arm amplification	nagABE-C-F	TTGGTGACAAAACTCACAATCTGCTTTATGCCTGATGCGACGC
	nagABE-C-R	TGCATCCAACGCGTTGGGAGCTCTCCGGAGCTGATCAAAATAATCGG
nagB启动子扩增nagB promotor amplification	nagB-Pm-F	TGAGCTTGTCCGCCTGGTGTCATTATTCACCTCAATAAGTAAAATGTA
	nagB-Pm-R	CAAGCGTCGCATCAGGCATAAAGCAGATTGTGAGTTTTGTCACC
T7启动子扩增 T7 promotor amplification	nagABE-T7-F	TTTGGTGACAAAACTCACAAGGATCGAGATCTCGATCCCGCGAA
	nagABE-T7-R	GCAACGATATATTTTTTTTCAGTCATGGTATATCTCCTTCTTAAAGTT
glpK基因扩增 glpK gene amplification	pichi-glpk-F	CTTTAAGAAGGAGATATACCATGGGAAAAGACTATACACCAC
	pichi-glpk-R	CGTCGCATCAGGCATAAAGCAGATTAAGCAGTGTCCTTAAGCCAGCCC
单质粒glpK基因整合glpK gene integration（single plasmid system）
	ABE-F1	GTCAGATCTCCTGCAGCGCCAGTGCTTTCGTTTTAGAGCTAGAAATAGCAAGTTAAAA- TAAGG
	ABE -R1	GCATATTCAGGTAGAAAACTGGTCCCGCTTTGTTACAGAATGC
	ABE-up-F1	GCGGGACCAGTTTTCTACCTGAATATGCGGCATGTAATGAATTTTGCCGCTGTC
	ABE-down-R1	TGTTACGCGTTTTTGTCATGGCTTTGGAGCTGATCAAAATAATCGGAG
	ABE-F2	CTCCAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTATAATCACGGC
	ABE-R2	CGAAAGCACTGGCGCTGCAGGAGATCTGACTCCATAACAGAGTACTC
突变株检测引物Detection primer for mutants
	zwf-JC-F	ACGGCACAAACACCGCAGGC
	zwf-JC-R	ACATGATCAAGCGTTGCCATTGC
	A-JC-F	TTATCCTTGTCTCGTTTCAGCGTTGGGTGGTGCG
	A-JC-R	GTTTGATCCACTCTTTATCAATC
	B-JC-F	CATCGCGCTCTCGATAGCCGTTAT
	B-JC-R	GCGCAAATGCCATGCGGCATGGA
	ABE-JC-R	TTCAACGCTGCGGTCGCGGTAC
	ABE-JC-F	GGTTCCGCGATGGACACGCACC