Mobile CRISPR-Cas9 based anti-phage system in <i>E. coli</i>

doi:10.1007/s11705-022-2141-7

Front. Chem. Sci. Eng.

2022, Vol. 16

Issue (8) : 1281-1289 https://doi.org/10.1007/s11705-022-2141-7

RESEARCH ARTICLE

Mobile CRISPR-Cas9 based anti-phage system in E. coli

Zhou Cao^1,², Yuxin Ma^1,², Bin Jia^1,²(

), Ying-Jin Yuan^1,²

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

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Abstract

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.

Keywords phage infections anti-phage CRISPR-Cas9 conjugative transfer synthetic biology

Corresponding Author(s): Bin Jia

Online First Date: 25 February 2022 Issue Date: 02 August 2022

Cite this article:

Zhou Cao,Yuxin Ma,Bin Jia, et al. Mobile CRISPR-Cas9 based anti-phage system in E. coli[J]. Front. Chem. Sci. Eng., 2022, 16(8): 1281-1289.

URL:

https://academic.hep.com.cn/fcse/EN/10.1007/s11705-022-2141-7
https://academic.hep.com.cn/fcse/EN/Y2022/V16/I8/1281

Fig.1 A schematic representation of MCBAS for blocking phage infection. The MCBAS consist of CRISPR-Cas9 for cleavage of phage DNA and origin of transfer (oriT) for conjugation. To clear phage DNA in cells, the plasmids were delivered to recipient cells infected by phage via conjugation to remove phage DNA. To prevent phage infection, the CRISPR-Cas9 plasmids were delivered to recipient cells before phage infection via conjugation to prevent phage DNA replication.

Fig.2 Design of MKR phage. (a) Design of the MKR phage used to infect the E. coli XL1-Blue strain; (b) fluorescence images of the E. coli XL1-Blue strain infected by the MKR phage in the bright field (left) and red channel (right); (c) the MKR phage infection rate of the E. coli XL1-Blue strain after adding 2 µL MKR phage (MOI (multiplicity of infection) = 1:500, error bars represent standard?deviations (SDs) from three independent experiments).

Fig.3 MCBAS clears phage DNA in E. coli. (a) A schematic representation of MCBAS clearing MKR phage DNA in E. coli. (b) The MCBAS containing oriT for conjugative transfer. (c) The conjugation assay between donor cells (JM109) and recipient cells (XL10-Blue). JM109 with pGM0 (Cm), XL1-Blue with pUC19 (Amp) and mixture of the two strains were dropped to the LB agar plate with Cm and Amp antibiotics. (d) The MKR phage infection rate of the E. coli XL1-Blue strain infected with the MKR phage over time after transformation with pGM1 and pGM2 (?Error bars represent SDs from three independent experiments). (e) Fluorescence images of the E. coli XL1-Blue strain carrying pGM1 and pGM2 for phage DNA clearance at 12 h in the bright field (left), red channel (middle) and green channel (right).

Fig.4 MCBAS prevents phage infection of E. coli. (a) Schematic representation of MCBAS preventing MKR phage infection. The MKR phage infection rates of the E. coli XL1-Blue strain carrying pGM1 and pGM2 over time are shown in (b), (c), (d) and (e) when the MOI was adjusted to 1:500, 1:100, 1:20 and 1:10, respectively (Error bars represent SDs from three independent experiments).

Fig.5 Sequence verification of escaped phages. (a) Alignment of the escape mutant sequences of 10 different infected E. coli pGM1 strains; (b) alignment of the escape mutant sequences of 10 different infected E. coli pGM2 strains.

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