Jinming Lei¹, Chenfeng He¹, Baoyao Huang¹, Hailing Zhou¹, Jiaxin Li¹, Ruoran Qiu¹, Zuyan Fan¹, Zihua He¹, Xiaoxuan Long¹, Chang Liu¹, Yi Zhong¹, Wencui Ma¹, Wenjia Liu¹, Yan Gao¹, Yanmei Liu², Rundong Long², Yanrui Ye³
¹iGEM Student Team Member, ²iGEM Team Mentor, ³iGEM Team Primary PI, §Faculty Sponsor,School of Biology and Biological Engineering, South China University of Technology, Guangzhou, China
Abstract
In this project, we show that the genome of the lytic P. Aeruginosa phage vB_PaeM_SCUT-S1 (S1) can be engineered by a CRISPR-Cas editing strategy based on a two-plasmid system in which the first plasmid expresses a Cas nuclease and a recombinase (λ-red), the second plasmid harbors a crRNA cassette and a repair template. The strategy showed excellent efficiency and after editing, all the plaques tested contained the recombinant phages with the desired mutation.
Briefly, we constructed two phage mutants in our project. The first mutant T_S1 was obtained by inserting relE gene within the ORF73 of S1, a hypothetical protein, while RelE has great inhibitory effect on bacterial growth in vitro and in vivo. For further biosafety consideration, we constructed a lysis-deficient phage (LyDT_S1) expressing the antibacterial toxin RelE. The mutant was obtained by replacing the gene of holing, a lysis-promoting phage toxin, with the relE gene, which can still inhibit bacterial growth through the expression of RelE but cannot induce cell lysis because the holin gene is eliminated. This is expected to reduce the amount of endotoxins released from the pathogen bacteria, with a corresponding reduction of systemic cytokine response and inflammation during bacterial infection. In lysis kinetics for characterization of bacteriostasis effect, high MOI (multiplicity of infection) group of mutants indicates that bacteria do not develop resistance to phages. It might help solve one of the main questions regarding phage therapy，the possible rapid emergence of phage - resistant bacterial variants, which could impede favourable treatment outcomes.
This strategy of replacing the expression of holin with that of a toxin is a demonstrative example of the potential of this editing system based on CRISPR-Cas and λ-red plasmids, which we hope will serve as an efficient tool for editing the hard-to-engineer phages genomes, to better understand phage-host interactions and pave the way for the modular design of phages.
Team:SCUT China/Poster
Poster: SCUT_China
Engineering & Test of the lytic P. Aeruginosa phage S1 using CRISPR-Cas & λ-red
Project goals
One of bioeconomy, societal challenge is to eradicate existing and emerging infectious diseases. The engineering biology objective related is to develop tools treat microbial infections, overcome antimicrobial-resistance, and reduce the dependence upon antibiotics in human, pets, livestock, and other animal populations.
Our project is a grain of sand in the road which leads to develop evolvable therapies and rapid design and synthesis of customized, targeted therapeutics for inhibiting pathogenic cell growth.
Our team initially aim to focus on antibiotic resistant bacterial pathogens. Advancements within synthetic biology technologies have opened up new avenues towards the search for the alternatives for commercial antibiotics. Phage has a high degree of host specificity over broad-spectrum antibiotics without hazarding the host and its commensal microbiota in the era of precision medicine. Therefore, we developed an efficient phage genome editing strategy, after editing, all the plaques tested contained the recombinant phages with the desired mutation. We created two mutants and the lysis kinetics demonstrated that bacteria do not develop resistance to phage mutants. It might help solve one of the main questions regarding phage therapy，the possible rapid emergence of phage - resistant bacterial variants, which could impede favourable treatment outcomes.
We discussed detailed and comprehensive proposed implementation，containing investigation of existing (engineered) phage products, collection of relevant laws & regulations on phage drugs to stimulate the development of products and discussion about the Obstacles & Challenges to phage treatment and corresponding suggestions.
We diversified channels and developed science communication content for different target audience to address the public’s confusion about antibiotics and help develop the habit of proper medication.
Our project is a grain of sand in the road which leads to develop evolvable therapies and rapid design and synthesis of customized, targeted therapeutics for inhibiting pathogenic cell growth.
Our team initially aim to focus on antibiotic resistant bacterial pathogens. Advancements within synthetic biology technologies have opened up new avenues towards the search for the alternatives for commercial antibiotics. Phage has a high degree of host specificity over broad-spectrum antibiotics without hazarding the host and its commensal microbiota in the era of precision medicine. Therefore, we developed an efficient phage genome editing strategy, after editing, all the plaques tested contained the recombinant phages with the desired mutation. We created two mutants and the lysis kinetics demonstrated that bacteria do not develop resistance to phage mutants. It might help solve one of the main questions regarding phage therapy，the possible rapid emergence of phage - resistant bacterial variants, which could impede favourable treatment outcomes.
We discussed detailed and comprehensive proposed implementation，containing investigation of existing (engineered) phage products, collection of relevant laws & regulations on phage drugs to stimulate the development of products and discussion about the Obstacles & Challenges to phage treatment and corresponding suggestions.
We diversified channels and developed science communication content for different target audience to address the public’s confusion about antibiotics and help develop the habit of proper medication.
Motivation
We identified 4 areas of synthetic biology: Building, Optimizing, Test and Model.
Building : we show that the genome of the lytic P. Aeruginosa phage vB_PaeM_SCUT-S1 (S1) can be engineered by a CRISPR-Cas editing strategy based on a two-plasmid system in which the first plasmid expresses a Cas nuclease and a recombinase (λ-red), the second plasmid harbors a crRNA cassette and a repair template.
Optimizing: Editing sites: ORF73 → ORF 53 CRISPR Cas: SpCas9 → FnCpf1
Test: one step growth curve & lysis kinetics to determine the bacteriostatic effect of phage mutant
Model
Optimizing: Editing sites: ORF73 → ORF 53 CRISPR Cas: SpCas9 → FnCpf1
Test: one step growth curve & lysis kinetics to determine the bacteriostatic effect of phage mutant
Model
Building
We show that the genome of the lytic P. Aeruginosa phage vB_PaeM_SCUT-S1 (S1) can be engineered by a CRISPR-Cas editing strategy based on a two-plasmid system in which the first plasmid expresses a Cas nuclease and a recombinase (λ-red), the second plasmid harbors a crRNA cassette and a repair template.
The editing mechanism follows 4 general steps: spacer acquisition, biosynthesis of crRNAs, interference and homologous recombination. In the spacer acquisition step, a bacterial cell(BIM, bacteriophage insensitive mutant) acquires a new repeat-spacer unit in CRISPR locus following a phage challenge. The new spacer is acquired from the invading DNA through the involvement of protospacer adjacent motif(PAM)， and the CRISPR locus is processed with the help of trans-acting RNA(tracrRNA) and the host RNase Ⅲ to produce crRNA. Cas protein and crRNA complex guide and cleave the invading phage, and the break at the protospacer site can be repaired and recombined with a donor DNA to generate mutants of interest. The short time window of the lytic phage life cycle may limit the application of CRISPR-Cas to edit phage genome, so we accomplish through λ-red promoted homologous recombination with high efficiency. The strategy showed excellent efficiency and after editing, all the plaques tested contained the recombinant phages with the desired mutation.
We believed that this approach can be easily adapted to engineer any lytic phage infecting other bacterial species. As interest in the use of lytic phages for biocontrol and therapy purposes increases, we offer a new tool to modify a phage of interest to maximize its efficacy.
Optimizing
Optimizing1:
We constructed two phage mutants in our project. The first mutant T_S1 was obtained by inserting relE gene within the ORF73 of S1, a hypothetical protein, while RelE inhibits protein synthesis by cleaving mRNA codons on the ribosomal A site in a sequence specific way with preference for the stop codon UAG and has great inhibitory effect on bacterial growth in vitro and in vivo.
Furthermore, the clinical application of phage therapy faces challenge, among them the innate and adaptive bacterial resistance, immune reactions stimulated by bacterial lysis and so on. The use of nonreplicating modified or lysis-deficient phage for the delivery of genes encoding proteins toxic to bacterial pathogens may be a better solution towards undesired side effects of phage therapy resulted from endotoxin release. For biosafety consideration, we constructed a lysis-deficient phage (LyDT_S1) expressing the antibacterial toxin RelE. The second mutant was obtained by replacing the gene of holing, a lysis-promoting phage toxin, with the relE gene, which still inhibit bacterial growth through the expression of RelE but cannot induce cell lysis because the holin gene is eliminated. This is expected to reduce the amount of endotoxins released from the pathogen bacteria, with a corresponding reduction of systemic cytokine response and inflammation during bacterial infection.
Optimizing2:
For efficient editing, we constructed 7 recombinant plasmids each containing a around 23-t spacer sequence and transformed into PAO1(Table 1 some of the data shown). The spacer were inserted into the plasmid pYM005, and kept under the control of the J23119 promotor which constitutively expresses the corresponding crRNA. If the crRNA-tracrRNA-Cas9/Cpf1 complex is functional, it would cleave at the protospacer sequence of S1 and lead to a loss of plaque forming ability. The editing efficiency is inversely to the restriction of phage S1, determined by the efficiency of plating (EOP). We found that the EOP of Cpf1-crRNA（73-T9） was much lower than Cas-gRNA(73-T1) when the first mutant T_S1 was constructed. To improve editing efficiency, we used Fn Cpf1-crRNA（53-T1） to construct the second mutant LyDT_S1.
So far, nearly almost applications have been based on Cas9, the Cas effector protein of the type II CRISPR-Cas system. FnCpf1, derived from a type V CRISPR system, has three characteristics different from Cas9: first, Cpf1-associated CRISPR arrays are processed into mature crRNA without additional trans-activating crRNA. Secondly, the protospacer adjacent motif (PAM) following the target DNA for Cas9 is G-rich, while Cpf1-crRNA complex cleave target DNA proceeded by a short T-rich PAM. Thirdly，Cpf1 cleaves DNA via a staggered double strand break rather than blunt end. CRISPR-Cpf1-based genome editing provides a highly efficient tool for genetic engineering of bacteria that cannot utilize the CRISPR-SpCas9 system. Herein, we showed that complete gene replacement can be engineered in S1 genome using CRISPR- FnCpf1.
In summary, these are the dual-plasmid systems used to construct phage mutants.
We constructed two phage mutants in our project. The first mutant T_S1 was obtained by inserting relE gene within the ORF73 of S1, a hypothetical protein, while RelE inhibits protein synthesis by cleaving mRNA codons on the ribosomal A site in a sequence specific way with preference for the stop codon UAG and has great inhibitory effect on bacterial growth in vitro and in vivo.
Furthermore, the clinical application of phage therapy faces challenge, among them the innate and adaptive bacterial resistance, immune reactions stimulated by bacterial lysis and so on. The use of nonreplicating modified or lysis-deficient phage for the delivery of genes encoding proteins toxic to bacterial pathogens may be a better solution towards undesired side effects of phage therapy resulted from endotoxin release. For biosafety consideration, we constructed a lysis-deficient phage (LyDT_S1) expressing the antibacterial toxin RelE. The second mutant was obtained by replacing the gene of holing, a lysis-promoting phage toxin, with the relE gene, which still inhibit bacterial growth through the expression of RelE but cannot induce cell lysis because the holin gene is eliminated. This is expected to reduce the amount of endotoxins released from the pathogen bacteria, with a corresponding reduction of systemic cytokine response and inflammation during bacterial infection.
Optimizing2:
For efficient editing, we constructed 7 recombinant plasmids each containing a around 23-t spacer sequence and transformed into PAO1(Table 1 some of the data shown). The spacer were inserted into the plasmid pYM005, and kept under the control of the J23119 promotor which constitutively expresses the corresponding crRNA. If the crRNA-tracrRNA-Cas9/Cpf1 complex is functional, it would cleave at the protospacer sequence of S1 and lead to a loss of plaque forming ability. The editing efficiency is inversely to the restriction of phage S1, determined by the efficiency of plating (EOP). We found that the EOP of Cpf1-crRNA（73-T9） was much lower than Cas-gRNA(73-T1) when the first mutant T_S1 was constructed. To improve editing efficiency, we used Fn Cpf1-crRNA（53-T1） to construct the second mutant LyDT_S1.
So far, nearly almost applications have been based on Cas9, the Cas effector protein of the type II CRISPR-Cas system. FnCpf1, derived from a type V CRISPR system, has three characteristics different from Cas9: first, Cpf1-associated CRISPR arrays are processed into mature crRNA without additional trans-activating crRNA. Secondly, the protospacer adjacent motif (PAM) following the target DNA for Cas9 is G-rich, while Cpf1-crRNA complex cleave target DNA proceeded by a short T-rich PAM. Thirdly，Cpf1 cleaves DNA via a staggered double strand break rather than blunt end. CRISPR-Cpf1-based genome editing provides a highly efficient tool for genetic engineering of bacteria that cannot utilize the CRISPR-SpCas9 system. Herein, we showed that complete gene replacement can be engineered in S1 genome using CRISPR- FnCpf1.
In summary, these are the dual-plasmid systems used to construct phage mutants.
Test
For T_S1, the latent period is 50min,the burst size is 42 pfu/infected cell substantially less than 134 pfu/infected cell of S1（Figure 5.a）; For LyDT_S1 & WT S1, latent period is 40min similar to WT S1, the burst size is 116 pfu/infected cell （Figure 5.b）.
To determine the bacteriostatic effect of phage mutant, lysis kinetics were measured (Figure 6). High MOI (multiplicity of infection) group indicates that bacteria do not develop resistance to phages. It might help solve one of the main questions regarding phage therapy，the possible rapid emergence of phage - resistant bacterial variants, which could impede favourable treatment outcomes. Time lag effect observed in low MOI (0.01) group (Figure 6.b. c purple) different from wild type S1, we supposed that RelE globally regulates biological device (phage), systems (bacteria) and their interactions. RelE displays codon-specific cleavage of mRNAs in the ribosomal A site and phage plunders host transcriptional translation apparatus for replication. The time delay effect occurs because more progeny phages are produced by multiplication in order to effectively lyse the bacteria. MOI (0.01 0.1 1) group of LyDT_S1& WT S1 mixture (Figure 6.c), phage resistance developed after 14 hours so that's characteristic of WT S1 (12h), probably because lysis-deficient mutants is underpowered.
To determine the bacteriostatic effect of phage mutant, lysis kinetics were measured (Figure 6). High MOI (multiplicity of infection) group indicates that bacteria do not develop resistance to phages. It might help solve one of the main questions regarding phage therapy，the possible rapid emergence of phage - resistant bacterial variants, which could impede favourable treatment outcomes. Time lag effect observed in low MOI (0.01) group (Figure 6.b. c purple) different from wild type S1, we supposed that RelE globally regulates biological device (phage), systems (bacteria) and their interactions. RelE displays codon-specific cleavage of mRNAs in the ribosomal A site and phage plunders host transcriptional translation apparatus for replication. The time delay effect occurs because more progeny phages are produced by multiplication in order to effectively lyse the bacteria. MOI (0.01 0.1 1) group of LyDT_S1& WT S1 mixture (Figure 6.c), phage resistance developed after 14 hours so that's characteristic of WT S1 (12h), probably because lysis-deficient mutants is underpowered.
MODEL
Current and future work
Human Practice
Acknowledgements
