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A Programmable CRISPR/Cas9-Based Phage

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A Programmable CRISPR/Cas9-Based Phage Liu et al. Microb Cell Fact (2020) 19:136 https://doi.org/10.1186/s12934-020-01393-2 Microbial Cell Factories RESEARCH Open Access A programmable CRISPR/Cas9-based phage defense system for Escherichia coli BL21(DE3) Li Liu1,2, Dongdong Zhao2,3, Lijun Ye2,3, Tao Zhan2,3, Bin Xiong2,3, Muzi Hu2,3, Changhao Bi2,3* and Xueli Zhang2,3* Abstract Escherichia coli BL21 is arguably the most popular host for industrial production of proteins, and industrial fermenta- tions are often plagued by phage infections. The CRISPR/Cas system is guided by a gRNA to cleave a specifc DNA cassette, which can be developed into a highly efcient programable phage defense system. In this work, we con- structed a CRISPR/Cas system targeting multiple positions on the genome of T7 phage and found that the system increased the BL21’s defense ability against phage infection. Furthermore, the targeted loci on phage genome played a critical role. For better control of expression of CRISPR/Cas9, various modes were tested, and the OD of the opti- mized strain BL21(pT7cas9, pT7-3gRNA, prfp) after 4 h of phage infection was signifcantly improved, reaching 2.0, which was similar to the control culture without phage infection. Although at later time points, the defensive ability of CRISPR/Cas9 systems were not as obvious as that at early time points. The viable cell count of the engineered strain in the presence of phage was only one order of magnitude lower than that of the strain with no infection, which further demonstrated the efectiveness of the CRISPR/Cas9 phage defense system. Finally, the engineered BL21 strain under phage attack expressed RFP protein at about 60% of the un-infected control, which was signifcantly higher than the parent BL21. In this work, we successfully constructed a programable CRISPR/Cas9 system to increase the ability of E. coli BL21’s to defend against phage infection, and created a resistant protein expression host. This work provides a simple and feasible strategy for protecting industrial E. coli strains against phage infection. Keywords: CRISPR/Cas9, E. coli BL21, Phage Introduction and associated proteins (Cas), is a molecular adaptive Escherichia coli is a major host for laboratory and indus- immune system of prokaryotes. Te sequences between trial production of proteins, among which the BL21 the arrays of CRISPR repeats are called spacers [3, 4]. series of strains are the most popular because they can When prokaryotes were infected by phages or other for- expresses heterologous proteins at high levels [1]. How- eign nucleotides, the CRISPR-Cas system takes up the ever, BL21 can be easy infected by bacteriophages which invading DNA and integrates it into CRISPR loci, which can lead to enormous economic losses in industrial pro- functions as a record or ‘memory’ [5]. Phage resistance duction [2], and there are no ideal solutions for this prob- specifcity is determined by the similarity between the lem to date [2]. spacer and phage sequences [6]. In some lytic phages, the Te CRISPR-Cas system, comprising clustered regu- products of early-injected genes can degrade host DNA, larly interspaced short palindromic repeats (CRISPR) inhibit the synthesis of host RNA and proteins, and pro- tect the phage DNA, after which the rest of the viral DNA is injected into the cell [7]. Te most spacers are acquired *Correspondence: [email protected]; [email protected] 2 Tianjin Institute of Industrial Biotechnology, Chinese Academy during DNA injection, in which early-injected genomic of Sciences, Tianjin 300308, P R China regions provide more efective adaptive immunity than Full list of author information is available at the end of the article late-injected genomic regions [8]. Tese loci within © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://crea- tivecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdo- main/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Liu et al. Microb Cell Fact (2020) 19:136 Page 2 of 9 CRISPR can be transcribed and processed into small in viruses are rare. Similarly, microbes employed in the RNAs guiding Cas to cleave the phage chromosome [9]. fermentation industry often sufer from various bacte- Wild type E. coli MG1655 strains have a CRISPR-Cas3 riophages, which makes a programmable CRISPR/Cas9- system, which belongs to type I CRISPR-Cas, comprised based phage defense system highly desirable to improve of CRISPR, Cascades (CRISPR associated complex for the consistency of the fermentation process and hence antiviral defense proteins) and Cas3 [10]. Te complete improve the process economics. CRISPR-Cas3 system can target diferent sites on the In this work, we chose the genomic loci within genes genomes of E. coli phages such as λ, T7, or T4, and lead ecoding T7 phage encoding tail tubular protein gp12, to a delay in the growth of phage progeny in the infected capsid assembly protein and 3.8 protein as the targets. culture [11]. However, E. coli BL21 does not carry any Te tail tubular protein gp12 forms the end of the tail CRISPR-Cas system according to its genomic sequence, of T7 phage, including conical tube, nozzle, and small and also according to predictions done using the CRIS- extensions below the fbers, which is important for PRs web server (http://crisp r.i2bc.paris -sacla y.fr/?tdsou viruses to inject their genomes into the bacterial cyto- rceta g=s_pcqq_aioms g), suggesting that the sensitiv- plasm [22]. Te capsid assembly protein is a predicted ity of BL21 to phages might due to its lack of adaptive protein for assembly of phage shell [23, 24]. And the 3.8 immunity. protein we selected is non-functional [24]. Our work On the other hand, CRISPR/Cas9, a type-II CRISPR- designs a simple and programable CRISPR/Cas9 system Cas system, is the most widely applied type due to its rela- to increase the ability of the widely used E. coli BL21 to tively simple functional mechanism compared with other defend itself against phage infection, producing a pro- CRISPR systems. CRISPR/Cas9 only requires a guide grammable resistant industrial host. RNA or crRNA/tracrRNA duplex with a Cas9 protein to function [3]. Te protospacer adjacent motifs (PAMs) Materials and methods are utilized to make CRISPR–Cas9 distinguish between Bacterial strains and culture conditions the self-target and non-self-target protospacers [12]. Te All strains used in this work (Additional fle 1: Table S1) CRISPR/Cas9 system was used to target human and plant are derived from E. coli BL21(DE3), and were cul- viruses [13]. Some CRISPR/Cas9 based approaches were tured at 37 °C or 30 °C in Luria–Bertani medium (LB, developed to defend against specifc human viruses as a 10 g/L, tryptone, 5 g/L yeast extract, 10 g/L NaCl) with potential antiviral strategy for clinical applications [14]. apramycin (50 μg/ml) and chloramphenicol (30 μg/ml) For example, an SaCas9/gRNA system was programed or kanamycin (50 μg/ml) or ampicillin (50 μg/ml) when to specifcally target HIV-1 provirus and suppress HIV-1 necessary. infection in Jurkat C11 cells [15]. CRISPR/Cas9 was also employed to target plant viruses, which provided a more Plasmid construction versatile viral resistance in important crops [16]. Class We used j5 DeviceEditor [25] to design DNA primers II CRISPR systems include Cas13a other than cas9 [17]. (Additional fle 1: Table S3). DNA fragments were PCR- CRISPR/Cas13a (C2c2) was used to engineer interference amplifed using prime star (Takara, Japan) or Phusion against the RNA-based Turnip Mosaic Virus (TuMV) in polymerase (NEB, USA). BsaI restriction endonuclease plants [18]. FnCas9 and C2c2 were programed to break and T4 ligase were purchased from Termo-Fisher Scien- down the genome of geminiviruses in plants, providing tifc (USA). All the plasmids (Table 1) used in this study resistance against virus infection by consistently reduc- were constructed via Golden Gate method [25]. ing virus accumulation [19]. CRISPR/Cas may provide We chose spacer sequences within the T7 phage tail novel and reliable approaches to control geminiviruses tubular protein gp12, capsid assembly protein, and 3.8 and other ssDNA viruses such as Banana bunchy top protein genes as the N20 sequences of gRNA (Addi- virus (BBTV). Recently, PAC-MAN (Prophylactic Anti- tional fle 1: Table S2). Te p3gRNA is assembled by three viral CRISPR in huMAN cells) was developed base on fragments from pTgRNA, pCgRNA and p3.8gRNA. Te CRISPR-Cas13 system, which can efectively degrade backbone containing TgRNA were amplifed with prim- RNA of SARS-CoV-2 sequences and live infuenza A ers 3gRNA-T-F and 3gRNA -T-R using pTgRNA as a tem- virus (IAV) in human lung epithelial cells [20]. PAC- plate. Te other two fragments are amplifed by primers MAN has the potential to become an important pan-cor- 3gRNA-C-F and 3gRNA-C-R and primers 3gRNA-H-F onavirus treatment by safe and efective respiratory tract and 3gRNA-H-R, respectively.
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