Efficient Engineering Of Bacteriophages Using Recombineering Combined With CRISPR/Cas Technology

Abuse of antibiotics leads to the increasingly bacterial drug resistance,which seriously threatens human and animal health.It is urgent to find an effective way to fight drug-resistant bacteria.The development of new antibiotics is slow and cannot fully meet the needs of clinical treatment.Phage is one of the promising alternatives to antibiotics.During the application of phages,it was found that the traditional model of screening of single natural phage for a specific pathogen was suitable to treat the infection of individuals but was incapable to deal with the intensively farmed animals,which was often infected with epidemic strains with varieties.Moreover,it is hard to screen lytic phages for a large number of pathogenic bacteria.Based on the established resources of natural phages,the next step would be construct synthetic phages with programmable specificity.Genetic codon expansion is a new technology of synthetic biology.It enables the incorporation of unnatural amino acids into proteins thereby enhancing their physical and chemical properties.To introduce unnatural amino acids into key proteins,the synthetic phage with wider antibacterial spectrum and higher antibacterial efficiency is expected in principle.To construct phage containing unnatural amino acids,genetic modification of the phage genome is required.The most effective way is to construct phagemid and modify it in E.coli or yeast,followed with phage rescue in the corresponding host.Therefore,this project focuses on phagemid construction,modification and rescue based on recombineering.We took the E.coli lambda phage as the model system,using ExoCET to direct clone the whole phage genomic DNA E.coli.Then the Red recombination system combined with ccdB counter-selection was applied for seamless modification.An AfⅢ restriction endoenzyme recognition site was introduced as a genetic marker to distinguish the rescued phage from the wild type.The phage was successfully rescued after the modified phagemid being electroporated into its host.Since the phagemid could be successful rescued,a serial of directional modifications of the phagemid were carried out.Firstly,the four stop codons were mutated from TAG to TGA,so that the Lambda phage containing not more the TAG codon.Therefore the TAG can be used to code an unnatural amino acid.In order to evaluate the efficiency and the TAG encoded unnatural amino acids in Lambda phage,the fluorescent reporter mNeonGreen was inserted into the nonessential B2 region.The rescued phage could express the mNeonGreen without affecting the lysis proliferation.In the next step,we mutated the 42aa of the mNeonGreen from GAT(Aspartate)to TAG for subsequent experiments.The bottleneck of functional genome research of phage is the difficulties of gene editing.The improvement of phage performance to against the bacteria is impeded as well.In this study,we took the lambda phage as the substrate to evaluate the efficiency of phage genome engineering by recombineering combined with CRISPR-Cas system.We firstly compared different systems in E.coli using a plasmid as substrate.It was found that the combination of Redαβ mediated recombineering together with the CRISPR-Cas3 system resulted the highest efficiency.Then,the recombination efficiency of Redαβ combined with CRISPR-Cas3 for lambda phage engineering was examined in two different experiments,either pre-infecting the E.coli with the phage or co-electroporating the prepared phage DNA.It was found that the CRISPR-Cas3 could significantly increase the efficiency of the recombinant phages by damaging the DNA of the wild type.The editing efficiency of co-electroporating the prepared phage DNA method was higher than that of pre-infecting the E.coli with the phage method.Stable expression of foreign protein in mammalian cells requires precise insertion of the expression cassette into well-characterized genomic locus.To efficient produce proteins in Chinese hamster ovary(CHO)cells,the recombinase-mediated cassette exchange(RMCE)technology was applied to establish a platform of stable cell line.Based on the experience of mouse transgenesis,the Hprt and Rosa26 loci were selected.The landing pad with recognition sites of Cre recombinase were inserted by CRISPR-Cas3 assisted gene targeting.Based on the cell lines with the RMCE landing pad,the DNA cassette could be efficiently integrated at the locus for stable expression of the foreign protein.Efficient expression is highly depended not only on the locus but also on the promoters.Therefore we constructed RMCE plasmids with a serial of promoters driving the mNeonGreen reporter.After integration of the expression cassettes by RMCE,the optimum combination of the loci and the promoter will be decided by evaluation of the mRNA and the florescent protein level.A new CHO cell line for stable expression will be established.