Pair your accounts.

Export articles to Mendeley

Get article recommendations from ACS based on references in your Mendeley library.

Pair your accounts.

Export articles to Mendeley

Get article recommendations from ACS based on references in your Mendeley library.

You’ve supercharged your research process with ACS and Mendeley!

Click to create an ACS ID

Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

Your Mendeley pairing has expired. Please reconnect
ACS Publications. Most Trusted. Most Cited. Most Read
My Activity

Figure 1Loading Img

Multiplexed CRISPR-Cpf1-Mediated Genome Editing in Clostridium difficile toward the Understanding of Pathogenesis of C. difficile Infection

  • Wei Hong
    Wei Hong
    Department of Biosystems Engineering, Auburn University, Auburn, Alabama 36849, United States
    Key Laboratory of Endemic and Ethnic Diseases of the Ministry of Education  and  Key Laboratory of Medical Molecular Biology, Guizhou Medical University, Ministry of Education, Guiyang, Guizhou Province, China
    More by Wei Hong
  • Jie Zhang
    Jie Zhang
    Department of Biosystems Engineering, Auburn University, Auburn, Alabama 36849, United States
    More by Jie Zhang
  • Guzhen Cui
    Guzhen Cui
    Key Laboratory of Medical Microbiology and Parasitology, Guizhou Medical University, Ministry of Education, Guiyang, Guizhou Province, China
    More by Guzhen Cui
  • Luxin Wang
    Luxin Wang
    Department of Animal Sciences, Auburn University, Auburn, Alabama 36849, United States
    More by Luxin Wang
  • , and 
  • Yi Wang*
    Yi Wang
    Department of Biosystems Engineering, Auburn University, Auburn, Alabama 36849, United States
    *E-mail: [email protected]
    More by Yi Wang
Cite this: ACS Synth. Biol. 2018, 7, 6, 1588–1600
Publication Date (Web):June 4, 2018
Copyright © 2018 American Chemical Society

    Article Views





    Other access options
    Supporting Info (4)»


    Abstract Image

    Clostridium difficile is often the primary cause of nosocomial diarrhea, leading to thousands of deaths annually worldwide. The availability of an efficient genome editing tool for C. difficile is essential to understanding its pathogenic mechanism and physiological behavior. Although CRISPR-Cas9 has been extensively employed for genome engineering in various organisms, large gene deletion and multiplex genome editing is still challenging in microorganisms with underdeveloped genetic engineering tools. Here, we describe a streamlined CRISPR-Cpf1-based toolkit to achieve precise deletions of fur, tetM, and ermB1/2 in C. difficile with high efficiencies. All of these genes are relevant to important phenotypes (including iron uptake, antibiotics resistance, and toxin production) as related to the pathogenesis of C. difficile infection (CDI). Furthermore, we were able to delete an extremely large locus of 49.2-kb comprising a phage genome (phiCD630-2) and realized multiplex genome editing in a single conjugation with high efficiencies (simultaneous deletion of cwp66 and tcdA). Our work highlighted the first application of CRISPR-Cpf1 for multiplexed genome editing and extremely large gene deletion in C. difficile, which are both crucial for understanding the pathogenic mechanism of C. difficile and developing strategies to fight against CDI. In addition, for the DNA cloning, we developed a one-step-assembly protocol along with a Python-based algorithm for automatic primer design, shortening the time for plasmid construction to half that of conventional procedures. The approaches we developed herein are easily and broadly applicable to other microorganisms. Our results provide valuable guidance for establishing CRISPR-Cpf1 as a versatile genome engineering tool in prokaryotic cells.

    Read this article

    To access this article, please review the available access options below.

    Get instant access

    Purchase Access

    Read this article for 48 hours. Check out below using your ACS ID or as a guest.


    Access through Your Institution

    You may have access to this article through your institution.

    Your institution does not have access to this content. You can change your affiliated institution below.

    Supporting Information

    Jump To

    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.8b00087.

    • Detail plasmid construction methods (PDF)

    • Tables of plasmids, strains, and primers used in this work (PDF)

    • Additional figures as described in the text (PDF)

    • Python script of OPF (ZIP)

    Terms & Conditions

    Electronic Supporting Information files are available without a subscription to ACS Web Editions. The American Chemical Society holds a copyright ownership interest in any copyrightable Supporting Information. Files available from the ACS website may be downloaded for personal use only. Users are not otherwise permitted to reproduce, republish, redistribute, or sell any Supporting Information from the ACS website, either in whole or in part, in either machine-readable form or any other form without permission from the American Chemical Society. For permission to reproduce, republish and redistribute this material, requesters must process their own requests via the RightsLink permission system. Information about how to use the RightsLink permission system can be found at

    Cited By

    This article is cited by 62 publications.

    1. William Rostain, Tom Zaplana, Magali Boutard, Chloé Baum, Sibylle Tabuteau, Mary Sanitha, Mohandass Ramya, Adam Guss, Laurence Ettwiller, Andrew C. Tolonen. Tuning of Gene Expression in Clostridium phytofermentans Using Synthetic Promoters and CRISPRi. ACS Synthetic Biology 2022, 11 (12) , 4077-4088.
    2. Linggang Zheng, Yang Tan, Yucan Hu, Juntao Shen, Zepeng Qu, Xianbo Chen, Chun Loong Ho, Elaine Lai-Han Leung, Wei Zhao, Lei Dai. CRISPR/Cas-Based Genome Editing for Human Gut Commensal Bacteroides Species. ACS Synthetic Biology 2022, 11 (1) , 464-472.
    3. Karin Krumbach, Christiane Katharina Sonntag, Lothar Eggeling, Jan Marienhagen. CRISPR/Cas12a Mediated Genome Editing To Introduce Amino Acid Substitutions into the Mechanosensitive Channel MscCG of Corynebacterium glutamicum. ACS Synthetic Biology 2019, 8 (12) , 2726-2734.
    4. Ran Zhao, Yanqiang Liu, Huan Zhang, Changsheng Chai, Jin Wang, Weihong Jiang, Yang Gu. CRISPR-Cas12a-Mediated Gene Deletion and Regulation in Clostridium ljungdahlii and Its Application in Carbon Flux Redirection in Synthesis Gas Fermentation. ACS Synthetic Biology 2019, 8 (10) , 2270-2279.
    5. Inés C. Cañadas, Daphne Groothuis, Maria Zygouropoulou, Raquel Rodrigues, Nigel P. Minton. RiboCas: A Universal CRISPR-Based Editing Tool for Clostridium. ACS Synthetic Biology 2019, 8 (6) , 1379-1390.
    6. Tian-Cai Niu, Gui-Ming Lin, Li-Rui Xie, Zi-Qian Wang, Wei-Yue Xing, Ju-Yuan Zhang, Cheng-Cai Zhang. Expanding the Potential of CRISPR-Cpf1-Based Genome Editing Technology in the Cyanobacterium Anabaena PCC 7120. ACS Synthetic Biology 2019, 8 (1) , 170-180.
    7. Hae Jun Kwon, Joungmin Lee, Soo Jae Kwon, Hyun Sook Lee. Development of a genetic engineering toolbox for syngas-utilizing acetogen Clostridium sp. AWRP. Microbial Cell Factories 2024, 23 (1)
    8. Joshua N. Brehm, Joseph A. Sorg. Theophylline-based control of repA on a Clostridioides difficile plasmid for use in allelic exchange. Anaerobe 2024, 88 , 102858.
    9. Jun Feng, Qingke Wang, Zhen Qin, Xiaolong Guo, Hongxin Fu, Shang‐Tian Yang, Jufang Wang. Development of inducible promoters for regulating gene expression in Clostridium tyrobutyricum for biobutanol production. Biotechnology and Bioengineering 2024, 121 (5) , 1518-1531.
    10. Jan Theys, Adam V. Patterson, Alexandra M. Mowday. Clostridium Bacteria: Harnessing Tumour Necrosis for Targeted Gene Delivery. Molecular Diagnosis & Therapy 2024, 11
    11. Linggang Zheng, Juntao Shen, Ruiyue Chen, Yucan Hu, Wei Zhao, Elaine Lai-Han Leung, Lei Dai. Genome engineering of the human gut microbiome. Journal of Genetics and Genomics 2024, 11
    12. Yanchao Zhang, Aleksandra M. Kubiak, Tom S. Bailey, Luuk Claessen, Philip Hittmeyer, Ludwig Dubois, Jan Theys, Philippe Lambin, . Development of a CRISPR-Cas12a system for efficient genome engineering in clostridia. Microbiology Spectrum 2023, 11 (6)
    13. Ziying Chen, Wenbing Jin, Alex Hoover, Yanjie Chao, Yanlei Ma. Decoding the microbiome: advances in genetic manipulation for gut bacteria. Trends in Microbiology 2023, 31 (11) , 1143-1161.
    14. Ali A. Rabaan, Mona A. Al Fares, Manar Almaghaslah, Tariq Alpakistany, Nawal A. Al Kaabi, Saleh A. Alshamrani, Ahmad A. Alshehri, Ibrahim Abdullah Almazni, Ahmed Saif, Abdulrahim R. Hakami, Faryal Khamis, Mubarak Alfaresi, Zainab Alsalem, Zainab A. Alsoliabi, Kawthar Amur Salim Al Amri, Amal K. Hassoueh, Ranjan K. Mohapatra, Kovy Arteaga-Livias, Mohammed Alissa. Application of CRISPR-Cas System to Mitigate Superbug Infections. Microorganisms 2023, 11 (10) , 2404.
    15. Wei Meng, Kai Qiao, Fan Liu, Xu Gao, Xuan Hu, Jia Liu, Yiyun Gao, Jianrong Zhu. Construction and application of a new CRISPR/Cas12a system in Stenotrophomonas AGS‐1 from aerobic granular sludge. Biotechnology Journal 2023, 18 (9)
    16. Muhammad Junaid, Krit Thirapanmethee, Piyatip Khuntayaporn, Mullika Traidej Chomnawang. CRISPR-Based Gene Editing in Acinetobacter baumannii to Combat Antimicrobial Resistance. Pharmaceuticals 2023, 16 (7) , 920.
    17. Cut Ulfah Nihayati Husaini, Rozieffa Roslan, Ahmad Bazli Ramzi, Abdullah Amru Indera Luthfi, Jian Ping Tan, Swee Su Lim, Gong Tao Ding, Jamaliah Md Jahim, Peer Mohamed Abdul. The CRISPR technology: A promising strategy for improving dark fermentative biohydrogen production using Clostridium spp.. International Journal of Hydrogen Energy 2023, 48 (61) , 23498-23515.
    18. Jack Arnold, Joshua Glazier, Mark Mimee, . Genetic Engineering of Resident Bacteria in the Gut Microbiome. Journal of Bacteriology 2023, 93
    19. Nicole D. Marino. Phage Against the Machine: Discovery and Mechanism of Type V Anti-CRISPRs. Journal of Molecular Biology 2023, 435 (7) , 168054.
    20. Duodong Wang, Mengcheng You, Zili Qiu, Ping Li, Mingqiang Qiao, Chenggang Xu. Development of an efficient ClosTron system for gene disruption in Ruminiclostridium papyrosolvens. Applied Microbiology and Biotechnology 2023, 107 (5-6) , 1801-1812.
    21. Raghul Senthilnathan, Ilamathi Ilangovan, Milind Kunale, Nalini Easwaran, Siva Ramamoorthy, Ashokkumar Veeramuthu, Gothandam Kodiveri Muthukaliannan. An update on CRISPR-Cas12 as a versatile tool in genome editing. Molecular Biology Reports 2023, 50 (3) , 2865-2881.
    22. Mao Chen, Yuhuan Huang, Yudi Zheng, Bo Wu, Mingxiong He. The escape of CRISPR-mediated gene editing in Zymomonas mobilis. FEMS Microbiology Letters 2023, 370
    23. Mariachiara Mengoli, Monica Barone, Marco Fabbrini, Federica D’Amico, Patrizia Brigidi, Silvia Turroni. Make It Less difficile: Understanding Genetic Evolution and Global Spread of Clostridioides difficile. Genes 2022, 13 (12) , 2200.
    24. Nicole LeBlanc, Trevor C. Charles. Bacterial genome reductions: Tools, applications, and challenges. Frontiers in Genome Editing 2022, 4
    25. Kondapalli Vamsi Krishna, Natarajan Bharathi, Shon George Shiju, Kuppusamy Alagesan Paari, Alok Malaviya. An updated review on advancement in fermentative production strategies for biobutanol using Clostridium spp.. Environmental Science and Pollution Research 2022, 29 (32) , 47988-48019.
    26. Dandan Jiang, Dandan Zhang, Shengnan Li, Yueting Liang, Qianwei Zhang, Xu Qin, Jinlan Gao, Jin‐Long Qiu. Highly efficient genome editing in Xanthomonas oryzae pv.  oryzae through repurposing the endogenous type I‐C CRISPR‐Cas system. Molecular Plant Pathology 2022, 23 (4) , 583-594.
    27. Qingshuai Zhou, Fengqin Rao, Zhenghong Chen, Yumei Cheng, Qifang Zhang, Jie Zhang, Zhizhong Guan, Yan He, Wenfeng Yu, Guzhen Cui, Xiaolan Qi, Wei Hong, . The cwp66 Gene Affects Cell Adhesion, Stress Tolerance, and Antibiotic Resistance in Clostridioides difficile. Microbiology Spectrum 2022,
    28. Michelle J. Chua, James Collins, , Robert Shields. Rapid, Efficient, and Cost-Effective Gene Editing of Enterococcus faecium with CRISPR-Cas12a. Microbiology Spectrum 2022, 10 (1)
    29. Wen-Bing Jin, Ting-Ting Li, Da Huo, Sophia Qu, Xin V. Li, Mohammad Arifuzzaman, Svetlana F. Lima, Hui-Qing Shi, Aolin Wang, Gregory G. Putzel, Randy S. Longman, David Artis, Chun-Jun Guo. Genetic manipulation of gut microbes enables single-gene interrogation in a complex microbiome. Cell 2022, 185 (3) , 547-562.e22.
    30. Duolong Zhu, Shaohui Wang, Xingmin Sun. FliW and CsrA Govern Flagellin (FliC) Synthesis and Play Pleiotropic Roles in Virulence and Physiology of Clostridioides difficile R20291. Frontiers in Microbiology 2021, 12
    31. Jorge Monteford, Terry W. Bilverstone, Patrick Ingle, Sheryl Philip, Sarah A. Kuehne, Nigel P. Minton. What's a SNP between friends: The lineage of Clostridioides difficile R20291 can effect research outcomes. Anaerobe 2021, 71 , 102422.
    32. Duolong Zhu, Hiran Malinda Lamabadu Warnakulasuriya Patabendige, Brooke Rene Tomlinson, Shaohui Wang, Syed Hussain, Domenica Flores, Yongqun He, Lindsey N. Shaw, Xingmin Sun. Cwl0971, a novel peptidoglycan hydrolase, plays pleiotropic roles in Clostridioides difficile R20291. Environmental Microbiology 2021, 23 (9) , 5222-5238.
    33. Rochelle C. Joseph, Susan Q. Kelley, Nancy M. Kim, Nicholas R. Sandoval. Metabolic Engineering and the Synthetic Biology Toolbox for Clostridium. 2021, 611-651.
    34. Zhanglin Lin, Huanhuan Li, Lan He, Yanyun Jing, Marco Pistolozzi, Tingting Wang, Yanrui Ye. Efficient genome editing for Pseudomonas aeruginosa using CRISPR-Cas12a. Gene 2021, 790 , 145693.
    35. Wei Hong, Feng-qin Rao, Xing-xing Zhao, Zhen Yang Guo, Yu-mei Chen, Bing Wang, Zhi-zhong Guan, Xiao-lan Qi. An inexpensive anaerobic chamber for the genetic manipulation of strictly anaerobic bacteria. Anaerobe 2021, 69 , 102349.
    36. Tom Wilding-Steele, Quentin Ramette, Paul Jacottin, Philippe Soucaille. Improved CRISPR/Cas9 Tools for the Rapid Metabolic Engineering of Clostridium acetobutylicum. International Journal of Molecular Sciences 2021, 22 (7) , 3704.
    37. Meliawati Meliawati, Christoph Schilling, Jochen Schmid. Recent advances of Cas12a applications in bacteria. Applied Microbiology and Biotechnology 2021, 105 (8) , 2981-2990.
    38. Xiaoyu Liu, Li Lin, Lianchao Tang, Haihua Xie, Lingkai Gu, Xiujuan Lv, Changbao Liu, Junzhao Zhao, Ruzhi Deng, Yong Liu, Jia Qu, Feng Gu. Lb2Cas12a and its engineered variants mediate genome editing in human cells. The FASEB Journal 2021, 35 (4)
    39. Barbara Bourgade, Nigel P Minton, M Ahsanul Islam. Genetic and metabolic engineering challenges of C1-gas fermenting acetogenic chassis organisms. FEMS Microbiology Reviews 2021, 45 (2)
    40. Pablo Jiménez-Bonilla, Jun Feng, Shangjun Wang, Jie Zhang, Yifen Wang, David Blersch, Luz Estela de-Bashan, Philippe Gaillard, Liang Guo, Yi Wang, . Identification and Investigation of Autolysin Genes in Clostridium saccharoperbutylacetonicum Strain N1-4 for Enhanced Biobutanol Production. Applied and Environmental Microbiology 2021, 87 (7)
    41. Baisong Tong, Huina Dong, Yali Cui, Pingtao Jiang, Zhaoxia Jin, Dawei Zhang. The Versatile Type V CRISPR Effectors and Their Application Prospects. Frontiers in Cell and Developmental Biology 2021, 8
    42. Prarthana Mohanraju, Ioannis Mougiakos, Justin Albers, Megumu Mabuchi, Ryan T. Fuchs, Jennifer L. Curcuru, Richard van Kranenburg, G. Brett Robb, John van der Oost. Development of a Cas12a-Based Genome Editing Tool for Moderate Thermophiles. The CRISPR Journal 2021, 4 (1) , 82-91.
    43. Zhenquan Liu, Huina Dong, Yali Cui, Lina Cong, Dawei Zhang. Application of different types of CRISPR/Cas-based systems in bacteria. Microbial Cell Factories 2020, 19 (1)
    44. Wenliang Hao, Feiya Suo, Qiao Lin, Qiaoqing Chen, Li Zhou, Zhongmei Liu, Wenjing Cui, Zhemin Zhou. Design and Construction of Portable CRISPR-Cpf1-Mediated Genome Editing in Bacillus subtilis 168 Oriented Toward Multiple Utilities. Frontiers in Bioengineering and Biotechnology 2020, 8
    45. Jie Zhang, Wei Hong, Liang Guo, Yifen Wang, Yi Wang. Enhancing plasmid transformation efficiency and enabling CRISPR‐Cas9/Cpf1‐based genome editing in Clostridium tyrobutyricum. Biotechnology and Bioengineering 2020, 117 (9) , 2911-2917.
    46. Lei Cheng, Di Min, Ru‐Li He, Zhou‐Hua Cheng, Dong‐Feng Liu, Han‐Qing Yu. Developing a base‐editing system to expand the carbon source utilization spectra of Shewanella oneidensis MR‐1 for enhanced pollutant degradation. Biotechnology and Bioengineering 2020, 117 (8) , 2389-2400.
    47. Y. Jiang, Y. Fu, Z. Ren, H. Gou, C. Xu. Screening and application of inducible promoters in Ruminiclostridium papyrosolvens. Letters in Applied Microbiology 2020, 10
    48. Disha Bhattacharjee, Joseph A. Sorg, . Factors and Conditions That Impact Electroporation of Clostridioides difficile Strains. mSphere 2020, 5 (2)
    49. Seong Woo Kwon, Kuppusamy Alagesan Paari, Alok Malaviya, Yu-Sin Jang. Synthetic Biology Tools for Genome and Transcriptome Engineering of Solventogenic Clostridium. Frontiers in Bioengineering and Biotechnology 2020, 8
    50. Zhaowei Wu, Yujue Wang, Yifei Zhang, Weizhong Chen, Yu Wang, Quanjiang Ji. Strategies for Developing CRISPR‐Based Gene Editing Methods in Bacteria. Small Methods 2020, 4 (2)
    51. Patrick Ingle, Daphne Groothuis, Peter Rowe, He Huang, Alan Cockayne, Sarah A. Kuehne, Weihong Jiang, Yang Gu, Christopher M. Humphreys, Nigel P. Minton. Generation of a fully erythromycin-sensitive strain of Clostridioides difficile using a novel CRISPR-Cas9 genome editing system. Scientific Reports 2019, 9 (1)
    52. S. A. Kuehne, J. I. Rood, D. Lyras. Clostridial Genetics: Genetic Manipulation of the Pathogenic Clostridia. 2019, 927-939.
    53. Anna Maikova, Victor Kreis, Anaïs Boutserin, Konstantin Severinov, Olga Soutourina, . Using an Endogenous CRISPR-Cas System for Genome Editing in the Human Pathogen Clostridium difficile. Applied and Environmental Microbiology 2019, 85 (20)
    54. Justin M Vento, Nathan Crook, Chase L Beisel. Barriers to genome editing with CRISPR in bacteria. Journal of Industrial Microbiology and Biotechnology 2019, 46 (9-10) , 1327-1341.
    55. Kathleen N. McAllister, Joseph A. Sorg, . CRISPR Genome Editing Systems in the Genus Clostridium : a Timely Advancement. Journal of Bacteriology 2019, 201 (16)
    56. S. A. Kuehne, J. I. Rood, D. Lyras, , , , , , . Clostridial Genetics: Genetic Manipulation of the Pathogenic Clostridia. Microbiology Spectrum 2019, 7 (3)
    57. Zhen‐Hai Li, Min Liu, Xiao‐Mei Lyu, Feng‐Qing Wang, Dong‐Zhi Wei. CRISPR/Cpf1 facilitated large fragment deletion in Saccharomyces cerevisiae. Journal of Basic Microbiology 2018, 58 (12) , 1100-1104.
    58. Li Lin, Xiubin He, Tianyuan Zhao, Lingkai Gu, Yeqing Liu, Xiaoyu Liu, Hongyan Liu, Fayu Yang, Mengjun Tu, Lianchao Tang, Xianglian Ge, Changbao Liu, Junzhao Zhao, Zongming Song, Jia Qu, Feng Gu. Engineering the Direct Repeat Sequence of crRNA for Optimization of FnCpf1-Mediated Genome Editing in Human Cells. Molecular Therapy 2018, 26 (11) , 2650-2657.
    59. Kamil Charubin, R. Kyle Bennett, Alan G. Fast, Eleftherios T. Papoutsakis. Engineering Clostridium organisms as microbial cell-factories: challenges & opportunities. Metabolic Engineering 2018, 50 , 173-191.
    60. Ruilian Yao, Di Liu, Xiao Jia, Yuan Zheng, Wei Liu, Yi Xiao. CRISPR-Cas9/Cas12a biotechnology and application in bacteria. Synthetic and Systems Biotechnology 2018, 3 (3) , 135-149.
    61. Louis-Charles Fortier. Bacteriophages Contribute to Shaping Clostridioides (Clostridium) difficile Species. Frontiers in Microbiology 2018, 9
    62. Anna Maikova, Konstantin Severinov, Olga Soutourina. New Insights Into Functions and Possible Applications of Clostridium difficile CRISPR-Cas System. Frontiers in Microbiology 2018, 9