Mobile CRISPR-Cas9 based anti-phage system in E. coli
Zhou Cao1,2, Yuxin Ma1,2, Bin Jia1,2(), Ying-Jin Yuan1,2
1. Frontier Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin 300072, China 2. Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
Escherichia coli is one of the most important microbial cell factories, but infection by bacteriophages in the environment may have a huge impact on its application in industrial production. Here, we developed a mobile CRISPR-Cas9 based anti-phage system for bacteriophages defense in E. coli. Two conjugative plasmids pGM1 (phosphoglucomutase 1) and pGM2 carrying one and two guide RNAs, respectively, were designed to defend against a filamentous phage. The results showed that the pGM1 and pGM2 could decrease the phage infection rate to 1.6% and 0.2% respectively in infected cells. For preventing phage infection in E. coli, the pGM2 decreased the phage infection rate to 0.1%, while pGM1 failed to block phage infection. Sequence verification revealed that point mutations in protospacer or protospacer adjacent motif sequences of the phage genome caused loss of the defense function. These results support the potential application of MCBAS in E. coli cell factories to defend against phage infections.
. [J]. Frontiers of Chemical Science and Engineering, 2022, 16(8): 1281-1289.
Zhou Cao, Yuxin Ma, Bin Jia, Ying-Jin Yuan. Mobile CRISPR-Cas9 based anti-phage system in E. coli. Front. Chem. Sci. Eng., 2022, 16(8): 1281-1289.
J Li, P Neubauer. Escherichia coli as a cell factory for heterologous production of nonribosomal peptides and polyketides. New Biotechnology, 2014, 31(6): 579–585 https://doi.org/10.1016/j.nbt.2014.03.006
2
N A Baeshen, M N Baeshen, A Sheikh, R S Bora, M M M Ahmed, H A I Ramadan, K S Saini, E M Redwan. Cell factories for insulin production. Microbial Cell Factories, 2014, 13(1): 141 https://doi.org/10.1186/s12934-014-0141-0
3
Z Wang, J Sun, Q Yang, J Yang. Metabolic engineering Escherichia coli for the production of lycopene. Molecules (Basel, Switzerland), 2020, 25(14): 3136 https://doi.org/10.3390/molecules25143136
4
K Lemuth, K Steuer, C Albermann. Engineering of a plasmid-free Escherichia coli strain for improved in vivo biosynthesis of astaxanthin. Microbial Cell Factories, 2011, 10(1): 29 https://doi.org/10.1186/1475-2859-10-29
5
M Li, R Nian, M Xian, H Zhang. Metabolic engineering for the production of isoprene and isopentenol by Escherichia coli. Applied Microbiology and Biotechnology, 2018, 102(18): 7725–7738 https://doi.org/10.1007/s00253-018-9200-5
6
C Zhao, Y Zhang, Y Li. Production of fuels and chemicals from renewable resources using engineered Escherichia coli. Biotechnology Advances, 2019, 37(7): 107402 https://doi.org/10.1016/j.biotechadv.2019.06.001
7
H Wu, Z Fan, X Jiang, J Chen, G Chen. Enhanced production of polyhydroxybutyrate by multiple dividing E. coli. Microbial Cell Factories, 2016, 15(1): 128 https://doi.org/10.1186/s12934-016-0531-6
8
G Chen, X Jiang. Engineering bacteria for enhanced polyhydroxyalkanoates (PHA) biosynthesis. Synthetic and Systems Biotechnology, 2017, 2(3): 192–197 https://doi.org/10.1016/j.synbio.2017.09.001
9
X Liu, K Hua, D Liu, Z Wu, Y Wang, H Zhang, Z Deng, B A Pfeifer, M Jiang. Heterologous biosynthesis of type II polyketide products using E. coli. ACS Chemical Biology, 2020, 15(5): 1177–1183 https://doi.org/10.1021/acschembio.9b00827
10
Y Tang, M Wang, H Qin, X An, Z Guo, G Zhu, L Zhang, Y Chen. Deciphering the biosynthesis of TDP-β-L-oleandrose in avermectin. Journal of Natural Products, 2020, 83(10): 3199–3206 https://doi.org/10.1021/acs.jnatprod.0c00902
11
D T Jones, M Shirley, X Y Wu, S Keis. Bacteriophage infections in the industrial acetone butanol (AB) fermentation process. Journal of Molecular Microbiology and Biotechnology, 2000, 2(1): 21–26
12
S Chaturongakul, P Ounjai. Phage-host interplay: examples from tailed phages and Gram-negative bacterial pathogens. Frontiers in Microbiology, 2014, 5: 442 https://doi.org/10.3389/fmicb.2014.00442
13
S Kronheim, I M Daniel, Z Duan, S Hwang, A I Wong, I Mantel, J R Nodwell, K L Maxwell. A chemical defence against phage infection. Nature, 2018, 564(7735): 283–286 https://doi.org/10.1038/s41586-018-0767-x
H G Hampton, B N J Watson, P C Fineran. The arms race between bacteria and their phage foes. Nature, 2020, 577(7790): 327–336 https://doi.org/10.1038/s41586-019-1894-8
16
D Scholl, S Adhya, C Merril. Escherichia coli K1's capsule is a barrier to bacteriophage T7. Applied and Environmental Microbiology, 2005, 71(8): 4872–4874 https://doi.org/10.1128/AEM.71.8.4872-4874.2005
17
K Vasu, V Nagaraja. Diverse functions of restriction-modification systems in addition to cellular defense. Microbiology and Molecular Biology Reviews, 2013, 77(1): 53–72 https://doi.org/10.1128/MMBR.00044-12
18
M Pleska, C C Guet. Effects of mutations in phage restriction sites during escape from restriction- modification. Biology Letters, 2017, 13(12): 20170646 https://doi.org/10.1098/rsbl.2017.0646
19
Y Zhou, X Xu, Y Wei, Y Cheng, Y Guo, I Khudyakov, F Liu, P He, Z Song, Z Li, et al.. A widespread pathway for substitution of adenine by diaminopurine in phage genomes. Science, 2021, 372(6541): 512–516 https://doi.org/10.1126/science.abe4882
20
R Barrangou, C Fremaux, H Deveau, M Richards, P Boyaval, S Moineau, D A Romero, P Horvath. CRISPR provides acquired resistance against viruses in prokaryotes. Science, 2007, 315(5819): 1709–1712 https://doi.org/10.1126/science.1138140
21
G P Smith. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science, 1985, 228(4705): 1315–1317 https://doi.org/10.1126/science.4001944
22
K M Esvelt, J C Carlson, D R Liu. A system for the continuous directed evolution of biomolecules. Nature, 2011, 472(7344): 499–550 https://doi.org/10.1038/nature09929
23
J C Carlson, A H Badran, N D A Guggiana, D R Liu. Negative selection and stringency modulation in phage-assisted continuous evolution. Nature Chemical Biology, 2014, 10(3): 216–222 https://doi.org/10.1038/nchembio.1453
24
D I Bryson, C Fan, L Guo, C Miller, D Soll, D R Liu. Continuous directed evolution of aminoacyl-tRNA synthetases. Nature Chemical Biology, 2018, 14(2): 186 https://doi.org/10.1038/nchembio0218-186
25
M de Leeuw, M Baron, O B David, A Kushmaro. Molecular insights into bacteriophage evolution toward its host. Viruses, 2020, 12(10): 1132 https://doi.org/10.3390/v12101132
26
R Chayot, B Montagne, D Mazel, M Ricchetti. An end-joining repair mechanism in Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America, 2010, 107(5): 2141–2146 https://doi.org/10.1073/pnas.0906355107
27
L Liu, D Zhao, L Ye, T Zhan, B Xiong, M Hu, C Bi, X Zhang. A programmable CRISPR/Cas9-based phage defense system for Escherichia coli BL21(DE3). Microbial Cell Factories, 2020, 19(1): 136 https://doi.org/10.1186/s12934-020-01393-2
28
H Dong, H Xiang, D Mu, D Wang, T Wang. Exploiting a conjugative CRISPR/Cas9 system to eliminate plasmid harbouring the mcr-1 gene from Escherichia coli. International Journal of Antimicrobial Agents, 2019, 53(1): 1–8 https://doi.org/10.1016/j.ijantimicag.2018.09.017
29
Z X Xie, B Z Li, L A Mitchell, Y Wu, X Qi, Z Jin, B Jia, X Wang, B X Zeng, H M Liu, et al.. “Perfect” designer chromosome V and behavior of a ring derivative. Science, 2017, 355(6329): 1046 https://doi.org/10.1126/science.aaf4704
30
Y Wu, B Z Li, M Zhao, L A Mitchell, Z X Xie, Q H Lin, X Wang, W H Xiao, Y Wang, X Zhou, et al.. Bug mapping and fitness testing of chemically synthesized chromosome X. Science, 2017, 355(6329): 1048 https://doi.org/10.1126/science.aaf4706
31
W G Chen, M Z Han, J T Zhou, Q Ge, P P Wang, X C Zhang, S Y Zhu, L F Song, Y J Yuan. An artificial chromosome for data storage. National Science Review, 2021, 8(5): 62–70 https://doi.org/10.1093/nsr/nwab028
32
L Wang, S Jiang, C Chao, W He, X Wu, F Wang, T Tong, X Zou, Z Li, J Luo, et al.. Synthetic genomics: from DNA synthesis to genome design. Angewandte Chemie International Edition, 2018, 57(7): 1748–1756 https://doi.org/10.1002/anie.201708741
33
J Cello, A V Paul, E Wimmer. Chemical synthesis of poliovirus cDNA: generation of infectious virus in the absence of natural template. Science, 2002, 297(5583): 1016–1018 https://doi.org/10.1126/science.1072266
34
H O Smith, C A H Iii, C Pfannkoch, J C Venter. Generating a synthetic genome by whole genome assembly: X174 bacteriophage from synthetic oligonucleotides. Proceedings of the National Academy of Sciences of the United States of America, 2003, 100(26): 15440–15445 https://doi.org/10.1073/pnas.2237126100
35
L Y Chan, S Kosuri, D. EndyRefactoring bacteriophage T7. Molecular Systems Biology, 2005, 1: 2005.0018
36
T Thao, F Labroussaa, N Ebert, P V Kovski, V Thiel. Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform. Nature, 2020, 582(7813): 561–565 https://doi.org/10.1038/s41586-020-2294-9
37
J Zhang, D Zhang, J Zhu, H Liu, S Liang, Y Luo. Efficient multiplex genome editing in Streptomyces via engineered CRISPR-Cas12a systems. Frontiers in Bioengineering and Biotechnology, 2020, 8: 726 https://doi.org/10.3389/fbioe.2020.00726
38
L Wang, H Wang, H Liu, Q Zhao, B Liu, L Wang, J Zhang, J Zhu, R Bao, Y Luo. Improved CRISPR-Cas12a-assisted one-pot DNA editing method enables seamless DNA editing. Biotechnology and Bioengineering, 2019, 116(6): 1463–1474 https://doi.org/10.1002/bit.26938
39
H Liu, L Wang, Y Luo. Blossom of CRISPR technologies and applications in disease treatment. Synthetic and Systems Biotechnology, 2018, 3(4): 217–228 https://doi.org/10.1016/j.synbio.2018.10.003
