Food and Fermentation Industries >

2019 , Vol. 45 >Issue 19: 1 - 7

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.021520

Research Report

Optimization of a CRISPR-Cpf1/ssDNA genome editing system for Corynebacterium glutamicum

  • WANG Ting ,
  • MA Hongkun ,
  • ZHAO Guihong ,
  • CAI Ningyun ,
  • ZHANG Dezhi ,
  • CHEN Ning
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  • 1(College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China)
    2(National and Local United Engineering Lab of Metabolic Control Fermentation Technology(Tianjin University of Science and Technology), Tianjin 300457, China)
    3(Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education(Tianjin University of Science and Technology), Tianjin 300457, China)
    4(Tianjin Engineering Research Center of Microbial Metabolism and Fermentation Process Control, Tianjin 300457, China)

Revised date: 2019-07-18

  Online published: 2019-11-15

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Abstract

Corynebacterium glutamicum is an important microbial cell factory, and genomic modification has become a primary way to regulate target metabolites. In order to improve the editing efficiency of site-directed mutation, an efficient and time-saving CRISPR-Cpf1/ssDNA genome editing system was established. The kanamycin resistance was used as a screening marker to construct a rigorous ssDNA model strain M-1 to verify and calculate editing efficiency. Firstly, strong promoter Ptuf was adopted to optimize cutting efficiency. Secondly, the recombination efficiency was optimized by recombinase RecT and reasonable length and addition amount of ssDNA. The results showed that under the dual action of promoter Ptuf and RecT, the gene editing efficiency increased to (80±5.7)% using the optimized combination of lagging strands (70 bp and 12.5 μg). Overall, the RecT-mediated CRISPR-Cpf1/ssDNA genome editing system can greatly accelerate the metabolic engineering modification of C. glutamicum.

Key words： CRISPR-Cpf1; ssDNA; Corynebacterium glutamicum; optimization; genome editing

Cite this article

WANG Ting , MA Hongkun , ZHAO Guihong , CAI Ningyun , ZHANG Dezhi , CHEN Ning . Optimization of a CRISPR-Cpf1/ssDNA genome editing system for Corynebacterium glutamicum[J]. Food and Fermentation Industries, 2019 , 45(19) : 1 -7 . DOI: 10.13995/j.cnki.11-1802/ts.021520

References

[1] 薛宁,李智祥,战俊杰,等.敲除aceA和gogat及过表达gltA对谷氨酸棒状杆菌GKGD合成α-酮戊二酸的影响[J].食品与发酵工业,2017,43(8):5-11.
[2] 张成林,龙辉,温冰,等.双底物指数流加和双阶段溶氧控制对谷氨酸棒状杆菌生产L-异亮氨酸的影响[J].食品与发酵工业,2014,40(4):1-6.
[3] IKEDA M,NAKAGAWA S.The Corynebacterium glutamicum genome: features and impacts on biotechnological processes[J].Applied Microbiology and Biotechnology,2003,62(2-3):99-109.
[4] NEVERA J,PTEK M.Tools for genetic manipulations in Corynebacterium glutamicum and their applications[J].Applied Microbiology and Biotechnology,2011,90(5):1 641.
[5] CHO J S,CHOI K R,PRABOWO C P S,et al.CRISPR/Cas9-coupled recombineering for metabolic engineering of Corynebacterium glutamicum[J].Metabolic Engineering,2017,42:157-167.
[6] FENG Z,MAO Y,XU N,et al.Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis[J].Proceedings of the National Academy of Sciences,2014,111(12):4 632-4 637.
[7] WU H,LI Y,MA Q, et al.Metabolic engineering of Escherichia coli for high-yield uridine production[J].Metabolic Engineering,2018,49:248-256.
[8] LAI S,WEI S,ZHAO B,et al.Generation of knock-in pigs carrying Oct4-tdTomato reporter through CRISPR/Cas9-mediated genome engineering[J].PloS One,2016,11(1):e0146562.
[9] KAMINSKI R,CHEN Y,FISCHER T,et al.Elimination of HIV-1 genomes from human T-lymphoid cells by CRISPR/Cas9 gene editing[J].Scientific Reports,2016,6:22 555.
[10] XU R,QIN R,LI H,et al.Generation of targeted mutant rice using a CRISPR-Cpf1 system[J].Plant Biotechnology Journal,2017,15(6):713-717.
[11] DONG D,REN K,QIU X,et al.The crystal structure of Cpf1 in complex with CRISPR RNA[J].Nature,2016,532(7 600):522.
[12] JIANG Y,QIAN F,YANG J,et al.CRISPR-Cpf1 assisted genome editing of Corynebacterium glutamicum[J].Nature Communications,2017,8:15179.
[13] LIU J,WANG Y,LU Y,et al.Development of a CRISPR/Cas9 genome editing toolbox for Corynebacterium glutamicum[J].Microbial Cell Factories,2017,16(1):205.
[14] HALL S D,KOLODNER R D.Homologous pairing and strand exchange promoted by the Escherichia coli RecT protein[J].Proceedings of the National Academy of Sciences,1994,91(8):3 205-3 209.
[15] 谭延振. 谷氨酸棒状杆菌基因敲除系统的构建[M].无锡:江南大学,2012.
[16] HUANG Y,LI L,XIE S,et al.Recombineering using RecET in Corynebacterium glutamicum ATCC14067 via a self-excisable cassette[J].Scientific Reports,2017,7(1):7916.
[17] CONG L,RAN F A,COX D,et al.Multiplex genome engineering using CRISPR/Cas systems[J].Science,2013,339(6121):819-823.
[18] JIANG W,BIKARD D,COX D,et al.RNA-guided editing of bacterial genomes using CRISPR-Cas systems[J].Nature Biotechnology,2013,31(3):233.
[19] PAQUET D,KWART D,CHEN A,et al.Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9[J].Nature,2016,533(7 601):125.
[20] PENG F,WANG X,SUN Y,et al.Efficient gene editing in Corynebacterium glutamicum using the CRISPR/Cas9 system[J].Microbial Cell Factories,2017,16(1):201.
[21] RICHARDSON C D,RAY G J,DEWITT M A,et al.Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA[J].Nature Biotechnology,2016,34(3):339.
[22] ZERBINI F,ZANELLA I,FRACCASCIA D,et al.Large scale validation of an efficient CRISPR/Cas-based multi gene editing protocol in Escherichia coli[J].Microbial Cell Factories,2017,16(1):68.
[23] TONG Y,CHARUSANTI P,ZHANG L,et al.CRISPR-Cas9 based engineering of actinomycetal genomes[J].ACS Synthetic Biology,2015,4(9):1 020-1 029.
[24] XU T,LI Y,SHI Z,et al.Efficient genome editing in Clostridium cellulolyticum via CRISPR-Cas9 nickase[J].Appl Environ Microbiol,2015,81(13):4 423-4 431.
[25] GARST A D,BASSALO M C,PINES G, et al.Genome-wide mapping of mutations at single-nucleotide resolution for protein,metabolic and genome engineering[J].Nature Biotechnology,2017,35(1):48.
[26] SHUMAN S,GLICKMAN M S.Bacterial DNA repair by non-homologous end joining[J].Nature Reviews Microbiology,2007,5(11):852-861.
[27] SHIRAISHI K,HANADA K,IWAKURA Y,et al.Roles of RecJ, RecO, and RecR in RecET-mediated illegitimate recombination in Escherichia coli[J].Journal of Bacteriology,2002,184(17):4 715-4 721.
[28] WU J,DENG A,SUN Q,et al.Bacterial genome editing via a designed toxin-antitoxin cassette[J].ACS Synthetic Biology,2017,7(3):822-831.
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