WO2023164934A1 - 高通量简化噬菌体基因组骨架的方法 - Google Patents

高通量简化噬菌体基因组骨架的方法 Download PDF

Info

Publication number
WO2023164934A1
WO2023164934A1 PCT/CN2022/079314 CN2022079314W WO2023164934A1 WO 2023164934 A1 WO2023164934 A1 WO 2023164934A1 CN 2022079314 W CN2022079314 W CN 2022079314W WO 2023164934 A1 WO2023164934 A1 WO 2023164934A1
Authority
WO
WIPO (PCT)
Prior art keywords
phage
cipgr
plasmid
library
mutant
Prior art date
Application number
PCT/CN2022/079314
Other languages
English (en)
French (fr)
Inventor
马迎飞
袁盛建
Original Assignee
深圳先进技术研究院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 深圳先进技术研究院 filed Critical 深圳先进技术研究院
Priority to PCT/CN2022/079314 priority Critical patent/WO2023164934A1/zh
Publication of WO2023164934A1 publication Critical patent/WO2023164934A1/zh

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof

Definitions

  • This application relates to the technical field of life sciences, in particular to a method for simplifying the skeleton of a phage genome with high throughput.
  • phages have problems such as host specificity and easy resistance, which directly affect the druggability of phages. Consequently, many synthetic biology efforts have been conducted on phages to overcome these limitations.
  • Tailless phages such as M13 and X174, have very compact and small genomes ( ⁇ 10 kb), which are relatively simple to encode a limited number of genes, making them easy to edit and understand the function of their genes.
  • the genomes of tailed phages are usually relatively large (14-500 kbp) and extremely diverse, which makes it challenging to obtain simplified genomes of tailed phages on a large scale.
  • One of the challenges is that there is no efficient way to identify non-essential genes of phages at the genome scale. For example, the 25 non-essential genes of the model bacteriophage T7 were obtained from the knowledge accumulated in a large number of studies in the last three to four decades.
  • bacteriophage The amplification of bacteriophage is completely dependent on its host, and its unique self-propagating properties make the methods widely used in bacteria (simplified genome methods widely used in microorganisms such as Escherichia coli, yeast, Bacillus and mycoplasma, such as homologous recombination, Tn5 mutations, etc.) may not be suitable for large-scale application in tailed phages.
  • simple genome methods widely used in microorganisms such as Escherichia coli, yeast, Bacillus and mycoplasma, such as homologous recombination, Tn5 mutations, etc.
  • phage genomes due to the highly diverse characteristics of phage genomes, it is difficult to obtain non-essential gene information through comparison of bioinformatics methods.
  • One of the purposes of the embodiments of the present application is to provide a high-throughput method for simplifying the backbone of the phage genome.
  • a high-throughput method for simplifying the phage genome backbone comprising the steps of:
  • CiPGr sequence library includes n CiPGr sequences, n is a natural number greater than or equal to 2, the CiPGr sequence comprises gRNA, and the gRNA of each CiPGr sequence is different;
  • CiPGr plasmid-bacteria library Transforming the CiPGr plasmid in the CiPGr plasmid library, screening transformed cells containing the CiPGr plasmid, extracting the CiPGr plasmid from the transformed cell; transforming the CiPGr plasmid into a host bacterium containing the spCas9 plasmid In, the CiPGr plasmid-bacteria library is obtained, and the CiPGr plasmid-bacteria library contains n kinds of CiPGr plasmid-bacteria;
  • CiPGr plasmid-bacteria library Cultivate the wild-type phage and the corresponding CiPGr plasmid-bacteria library in the culture medium, transfer the mutant phage product to a fresh CiPGr plasmid-bacteria library to continue culturing, and repeat iterative cultivation to generate a mutant phage library;
  • the steps of generating the mutant phage library are:
  • Phage library; part of the first-generation mutant phage library and the fresh CiPGr plasmid-bacteria library medium are continued to be cultivated to generate second-generation mutant phage products, and the second-generation mutant phage products are centrifuged to take the supernatant solution to obtain the second-generation mutant phage library; part of the second-generation mutant phage library and the fresh CiPGr plasmid-bacterial library are cultivated in a medium to generate the third-generation mutant phage product, and the third-generation mutant phage product is centrifuged. Mutate the phage product, and take the supernatant to obtain the third-generation mutant phage library; repeat this iterative culture step, so that the number of transfers reaches 300-600 times, and collect each mutant phage library.
  • the method further comprises screening for a single active mutant phage.
  • the screening for a single active mutant phage comprises:
  • the method also includes screening dominant mutant phages, the steps comprising:
  • mutant phages in the mutant phage library After mixing the mutant phages in the mutant phage library, co-culture with wild-type host cells, transfer to fresh wild-type host cells for re-cultivation, and repeat the step of "transferring to fresh wild-type host cells for re-culture" N-1 times to get TN mutant phage;
  • the method further comprises the determination of non-essential genes, quasi-essential genes and essential genes in the phage.
  • the determination of non-essential genes, quasi-essential genes and essential genes in the phage includes:
  • the deletable gene with a deletion frequency of ⁇ 5% is determined to be a quasi-essential gene of the phage, and the deletable gene with a deletion frequency of >5% is determined to be a non-essential gene of the phage.
  • Essential genes, genes for which no deletions were detected were identified as essential genes for the phage.
  • the CiPGr sequence further comprises a barcode, two homology arms, a promoter and primers.
  • the transformed cells containing the CiPGr plasmid are screened, and the CiPGr plasmid is extracted from the transformed cells, including:
  • the CiPGr plasmid in the CiPGr plasmid library is transformed into DH5 ⁇ Escherichia coli competent cells, transformed 2 to 10 times in parallel, and the transformed DH5 ⁇ Escherichia coli competent cells are collected; cultured on a petri dish containing the CiPGr plasmid, and the DH5 ⁇ Escherichia coli transformed cells containing the CiPGr plasmid were screened, and the CiPGr plasmid was extracted from the DH5 ⁇ Escherichia coli transformed cell.
  • PCR separation of the CiPGr sequences in the CiPGr sequence library according to different phages is also included.
  • the cycle condition of the PCR separation is:
  • the method of assembling the CiPGr sequence library with the pTarget plasmid backbone is as follows: after connecting the CiPGr sequence library and the pTarget plasmid backbone through Gibson assembly reaction, then purifying.
  • the step of culturing the wild-type phage and the corresponding CiPGr plasmid-bacteria library in the culture medium it also includes: adding the CiPGr plasmid-bacteria library to the LB medium, adding antibiotics and L-arabinose overnight culture.
  • the number of repetitions of the iterative culture is 300-600 times.
  • the titrated concentration of the mutant phage library is regularly determined; when the concentration of the obtained mutant phage is less than 10 5 /ml, wild-type host cells are used to cultivate the mutant phage library.
  • the phage is tailed phage, tailless phage or other eukaryotic viruses.
  • the phage is replaced by a eukaryotic virus
  • the host bacterium is replaced by a host cell
  • the CiPGr plasmid-bacteria is replaced by a CiPGr plasmid-cell.
  • the beneficial effect of the high-throughput method for simplifying the phage genome skeleton is: for the deletable gene of the phage, a CiPGr sequence library with different gRNAs in the CiPGr sequence is designed, and the CiPGr sequence is assembled with the pTarget plasmid backbone to form CiPGr plasmid (i.e. pTarget plasmid), the CiPGr plasmid is transformed into the host bacteria containing the spCas9 plasmid to obtain the CiPGr plasmid-bacterial library.
  • the host bacteria contain both the pTarget plasmid and the spCas9 plasmid, and different individual bacteria in the CiPGr plasmid-bacteria library may contain different pTarget plasmids.
  • gRNA guide RNA
  • the offspring of the mutant phage can amplify without the gene.
  • phages infect different CiPGr plasmid-bacteria, resulting in the deletion of different genes, thereby continuously producing mutant phages.
  • Mutant phages are continuously transferred to new double-plasmid-containing bacteria, namely CiPGr plasmid-bacteria, which may infect host bacteria containing different pTarget plasmids and delete different genes.
  • the continuous high-throughput deletion of genes from top to bottom will continuously accumulate gene deletions on the phage genome, and obtain a mutant phage library containing different deletion genes.
  • Figure 1 is a schematic flow diagram of the method for the high-throughput simplified phage genome backbone provided by the embodiment of the present application;
  • Fig. 2 is a schematic flow diagram of the method for the high-throughput simplified phage genome backbone provided by the embodiment of the present application;
  • Figure 3 is the phage deletable gene library provided in Example 1 of the present application.
  • Fig. 4 is the gel electrophoresis graph of gp4.7 and gp5.3 in the phage T7 genome in different transfer times among the mutants provided in Example 1 of the present application;
  • Fig. 5 is the phage plaque of the mutant phage T7 in the double-layer agar plate provided by Example 1 of the present application;
  • Fig. 6 is the bactericidal curve of mutant phages T7 and T4 provided in Example 1 of the present application to MG1655;
  • Fig. 7 is the bactericidal curve of the phage with stronger antibacterial ability than the wild type provided by Example 1 of the present application;
  • Fig. 8 is a gene map of the stronger mutant phage provided in Example 1 of the present application.
  • Phage synthetic biology can integrate functional genes or gene circuits into the phage genome to enhance its antibacterial activity and potential in phage therapy and other different bioengineering applications.
  • Such gene circuit integration can be challenging due to the limited space for DNA packaging of phage particles.
  • a phage's accessory gene can help a phage better adapt to a wide range of ecological settings, but it may not be necessary for a given situation. Deletion of these non-essential genes can create some space in the phage genome.
  • the removal of redundant genes from the phage genome will help advance the understanding of phage-host interaction patterns and phage physiology. This understanding could further facilitate the redesign of more robust phage genomes, paving the way for fundamental biological discoveries.
  • the embodiment of the present application provides a top-down method for continuous high-throughput deletion of genes to simplify the genome, that is, iteratively simplified phage genome method (CiPGr) relying on CRISPR-cas9.
  • the phage may be a tailed phage, or may be a tailless phage.
  • the method includes the following steps:
  • CiPGr sequence libraries of two or more phages, and assemble each CiPGr sequence library with the pTarget plasmid backbone to form a CiPGr plasmid library.
  • the CiPGr sequence library of the phage is designed for the potentially deletable gene sequence.
  • the CiPGr sequence library can be designed for two or more phages at the same time, wherein a CiPGr sequence library can be designed for one type of phage , it is also possible to design two CiPGr sequence libraries.
  • Each CiPGr sequence library includes n CiPGr sequences, the value of n depends on the number of potentially deletable genes in a phage, and n is a natural number greater than or equal to 2.
  • the CiPGr sequence includes at least a guide RNA (guide RNA, gRNA), and the gRNA of each CiPGr sequence is different, so as to match different deletable genes in a phage.
  • guide RNA guide RNA
  • gRNA guide RNA
  • the CiPGr sequence comprises a barcode, two homology arms, a promoter, gRNA and primers.
  • the barcode is used to distinguish different phage libraries, and the different phages are separated by barcode primer PCR;
  • the primer is a part of the gRNA sequence, which is used as the Gibson assembly site when "CiPGr sequence and pTarget plasmid backbone assembly" is performed for Gibson assembly .
  • CiPGr sequences were synthesized on a DNA chip to realize batch synthesis of CiPGr sequence libraries.
  • all designed CiPGr sequence libraries were synthesized in a 6000-strip size DNA chip.
  • the length of the CiPGr sequence is designed to be 200 bp, so as to facilitate the synthesis of the gene sequence on the DNA chip and reduce the synthesis cost.
  • the CiPGr sequence is 200 bp, in which the barcode is 20 bp, the two homology arms are 50 bp each, the promoter is 36 bp, the spacer is 20 bp, and the primer is 24 bp.
  • multiple CiPGr sequence libraries are split by PCR technology, that is, the CiPGr sequences in the CiPGr sequence library are separated by PCR according to different phages. Specifically, the CiPGr sequence library is separated by PCR through the primers and barcodes on the CiPGr sequence, and multiple CiPGr sequence libraries are split into a single CiPGr sequence library.
  • the cycle conditions for PCR separation are: 98°C, 2 minutes; 98°C, 10 s, 58°C, 20 s, 72°C, 6 s; 72°C, 10 minutes, stored at 4°C .
  • each CiPGr sequence library was assembled with the pTarget plasmid backbone
  • the CiPGr sequence in the CiPGr sequence library was assembled with the pTarget plasmid backbone to form a CiPGr plasmid
  • the CiPGr sequence in the CiPGr sequence library was assembled.
  • a CiPGr plasmid library was assembled with the pTarget plasmid backbone.
  • the pTarget plasmid backbone includes Cm R and Ori, wherein Cm R represents the chloramphenicol resistance gene, and the origin of Ori restricted replication.
  • the pTarget plasmid backbone can be amplified by PCR, and the pTarget plasmid backbone obtained by PCR amplification can be purified by gel electrophoresis.
  • the CiPGr sequence library is purified by gel electrophoresis before the CiPGr sequence library is assembled with the pTarget plasmid backbone.
  • each CiPGr sequence library and the pTarget plasmid backbone are ligated by Gibson assembly reaction, and then purified.
  • the ligation of each CiPGr library to the pTarget plasmid backbone was achieved through 4 parallel Gibson assembly reactions.
  • a pTarget plasmid containing the CiPGr sequence is obtained, and different single host cells may contain different pTarget plasmids.
  • CiPGr plasmid-bacteria library contains n kinds of CiPGr plasmid-bacteria.
  • the CiPGr plasmids in the CiPGr plasmid library are transformed to enrich the number of CiPGr plasmids.
  • the CiPGr plasmid in each CiPGr plasmid library is transformed into DH5 ⁇ Escherichia coli competent cells, and transformed 2 to 10 times in parallel, and the transformed DH5 ⁇ Escherichia coli competent cells are collected; the DH5 ⁇ Escherichia coli competent cells are plated Cultivate on a petri dish containing chloramphenicol and kanamycin, screen DH5 ⁇ Escherichia coli transformed cells containing CiPGr plasmid, and extract CiPGr plasmid from DH5 ⁇ Escherichia coli transformed cells.
  • the way of culturing DH5 ⁇ Escherichia coli competent cells on a petri dish containing chloramphenicol and kanamycin may be: culturing overnight at 37°C.
  • the CiPGr plasmid is transformed into a host bacterium containing the spCas9 plasmid to obtain a CiPGr plasmid-bacteria library, and the CiPGr plasmid-bacteria library contains n kinds of CiPGr plasmid-bacteria.
  • the host bacterium contains a two-plasmid system, namely the pTarget plasmid and the spCas9 plasmid.
  • the host bacterium containing the spCas9 plasmid is determined according to the type of phage.
  • the host bacteria containing the spCas9 plasmid can be Escherichia coli (Mg1655) containing the spCas9 plasmid and Salmonella typhimurium containing the spCas9 plasmid.
  • the obtained CiPGr plasmid-bacteria library can be stored at -80°C.
  • PCR against the CiPGr sequence library was performed and sequenced by HiSeq 2500 (Illumina). After removing low-quality data from the obtained raw sequencing data, compare the sequencing data to the designed CiPGr library sequence, and determine the 100% identical ratio of each CiPGr library sequence in the plasmid library.
  • the CiPGr plasmid-bacteria library was taken, added to the culture medium, and cultivated overnight.
  • the CiPGr plasmid-bacteria library is a CiPGr plasmid-bacteria library stored at -80°C
  • the CiPGr plasmid-bacteria library takes the CiPGr plasmid-bacteria library and add it to the culture medium.
  • the culture temperature is 37° C.
  • the medium can be LB medium.
  • antibiotics and L-arabinose are added to the culture medium.
  • CiPGr plasmid-bacteria were added to LB medium, and antibiotics and L-arabinose were added for overnight culture.
  • the wild-type phage and the corresponding CiPGr plasmid-bacterial library were cultured in the culture medium, the phage infected the corresponding CiPGr plasmid-bacterial library, and the phage DNA was injected into the cells of the corresponding CiPGr plasmid-bacterial library.
  • the gRNA encoded by pTarget in the CiPGr plasmid-bacteria guides the Cas9 nuclease encoded by spCas9 to bind to the target gene on the wild-type phage and make the target gene double-strand break. Repair by homologous recombination (HR) induced by homologous sequences, leading to deletion or disruption of phage genes.
  • HR homologous recombination
  • the offspring of the mutant phage can amplify without the gene.
  • Wild-type phages infect different CiPGr plasmid-bacteria in the CiPGr plasmid-bacteria library, resulting in the deletion of different genes, resulting in mutant phage products.
  • wild-type phage and the corresponding CiPGr plasmid-bacteria library were purified in the culture medium for the mutant phage products produced after the first culture to form the first-generation mutant phage library.
  • CiPGr plasmid-bacteria library refers to the CiPGr plasmid-bacteria that can be infected by wild-type phage.
  • the mutant phage product Take the mutant phage product and transfer it to a fresh CiPGr plasmid-bacteria library to continue culturing, so that the mutant phage product can be transferred to fresh bacteria containing double plasmids, which may infect bacteria containing different CiPGr plasmids and delete different genes.
  • the mutant phage products are continuously transferred to new double-plasmid-containing bacteria, ie, CiPGr plasmid-bacteria, which will continuously accumulate gene deletions on the wild-type phage genome.
  • the mutant strains with a growth advantage can produce more offspring, and eventually occupy a dominant position in the mutant population and become the dominant strain.
  • the number of repeated iterative cultures is 300-600 times, which facilitates the easy detection of non-essential genes, quasi-essential genes and essential genes.
  • the fresh CiPGr plasmid-bacterial library referred to in the examples of the present application refers to a CiPGr plasmid-bacterial library that has not been co-cultured with wild-type phage, and is not necessarily a freshly configured CiPGr plasmid-bacterial library.
  • the types of the CiPGr plasmid-bacterial library for repeated iterative cultivation, the CiPGr plasmid-bacterial library for repeated iterative cultivation, and the CiPGr plasmid-bacterial library for the first cultivation process are consistent .
  • the generation steps of the mutant phage library are:
  • the wild-type phage and the corresponding CiPGr plasmid-bacterial library were cultivated in the culture medium to generate the first-generation mutant phage product, the first-generation mutant phage product was centrifuged, and the supernatant was taken to obtain the first-generation mutant phage library;
  • the first-generation mutant phage library and fresh CiPGr plasmid-bacteria library medium were continuously cultivated to generate the second-generation mutant phage product, and the second-generation mutant phage product was centrifuged to obtain the supernatant to obtain the second-generation mutant phage library;
  • the second-generation mutant phage library and fresh CiPGr plasmid-bacteria library were cultured in the culture medium to generate the third-generation mutant phage product, centrifuged the third-generation mutant phage product, and took the supernatant to obtain the third-generation mutant phage library; repeat In this iterative culture step, the number of transfers reaches 300-600 times, and each mutant phage
  • mutant phage libraries In this case, the continuous transfer of mutant phage products to new double-plasmid-containing bacteria, CiPGr plasmid-bacteria, allowed the accumulation of gene deletions on the wild-type phage genome, resulting in the formation of mutant phages lacking different genes, various Mutant phages with gene deletions form a mutant phage library.
  • the various deleted genes in the mutant phage library form a set of deleteable genes.
  • the titer concentration of the mutant phage library is regularly determined; when the concentration of the obtained mutant phage is less than 10 5 /ml, wild-type host cells are used to cultivate the mutant phage library. Due to subculture, the number of mutant phages decreases. In this case, the number or concentration of mutant phages can be expanded by culturing wild-type host cells. Exemplarily, every 10 transfers, a titration concentration of the mutant phage library is determined.
  • the mutant phage library obtained after a certain number of transfers is sequenced to obtain the deletable gene information of the mutant phage.
  • all stored mutant phages can be sequenced, or the obtained mutant phages can be sequenced during the process of culturing the mutant phages.
  • PCR is used to monitor the deletion of a phage gene.
  • one ⁇ L of mutant phage supernatant is used as a template for PCR, and Ex Taq DNA polymerase (Takara, RR01AM) is used to amplify the target gene through appropriate primers, and the PCR method is performed by agarose gel electrophoresis and Sanger Sequencing verified deletion of the gene.
  • the DNA of the mutant phage library was extracted according to the manual of the phage DNA isolation kit (NORGEN, 46850), and DNA sequencing was performed using a HiSeq1500 sequencer.
  • the 20th, 30th, 40th and 50th transfer products of phage T7 and T4 are sequenced, and then every 50 to 100 transfers are sequenced.
  • the method provided in the examples of the present application can also screen a single active mutant phage.
  • screening for a single active mutant phage includes:
  • each phage has two libraries (8 CiPGr plasmid-bacteria libraries are formed in total).
  • 1 ⁇ 10 3 PFU of the mutant phage library was mixed with 300 ⁇ L of wild-type host cells and 10 mL of 0.7% LB agar in a tube, poured into a plate, and placed in the Incubate overnight at 37°C. Randomly pick 8 large and 8 small plaques, and then separate and purify.
  • the one-step growth curve determination method of phage is: after bacterial streaking, pick a single clone for overnight culture; freshly culture phage, measure the concentration, and dilute to 10 5 PFU for later use; preheat the medium, inoculate 10ml with 1% volume, A total of 3 tubes were cultured at 37°C for 2 hours, centrifuged at 7000g at room temperature, removed the supernatant, added 5ml of preheated medium, oscillated and mixed; added 0.2mmol/L CaCl, added 100 ⁇ L of phage, oscillated and mixed; stood at room temperature for 5 minutes Finally, add 30ml of preheated culture medium, shake and mix well; after cultivating for a certain period of time, take samples and continue to detect the concentration.
  • a simplified phage genome with activity can be obtained quickly and with high throughput, especially for new phages with a large number of unknown functional genes.
  • the phage mutants were isolated and single-clonal sequenced to obtain a simplified phage genome; and the phage mutant libraries were mixed together for competitive culture, and mutants with stronger properties than wild-type phages could be isolated.
  • the method provided in the examples of the present application can also screen dominant mutant phages, and the screening steps include:
  • mutant phages in the mutant phage library After mixing the mutant phages in the mutant phage library, co-culture with the corresponding wild-type host cells, transfer to fresh wild-type host cells for re-cultivation, and repeat the steps of "transfer to fresh wild-type host cells for re-culture" N-1 times, get TN mutant phage ((wherein, T represents transfer, N represents transfer number));
  • the mutant phage libraries of different transfers were mixed together, and the mixed mutant phage library (10 5 PFU) had the corresponding logarithmic growth (10 8 PFU ) wild-type host cells were co-cultured for 2 hours to obtain T1 mutant phages; part of the T1 mutant phages were transferred to fresh host cells for culture again, so that a total of 8 transfers were performed, and a total of 16-20 generations were amplified to obtain T8 mutant phages.
  • 10 3 PFU of T8 mutant phage 300 ⁇ L of wild-type host cells and 10 mL of 0.7% LB agar were added to a tube, mixed, poured into LB plates, and incubated overnight at 37°C. Pick 3 different plaques for purification.
  • 10 ⁇ L of overnight cultured host cells 200 ⁇ L of fresh LB medium were added to a 96-well plate, and a single plaque was purified and transferred to a 96-well plate and read on a microplate reader. cultured at 37°C, and the OD600 was detected every 10 min for 12 hours.
  • non-essential genes, quasi-essential genes and essential genes can be easily detected by performing metagenomic sequencing on the mutant phage library.
  • the method provided in the examples of the present application further includes the determination of non-essential genes, quasi-essential genes and essential genes in the phage.
  • the determination of non-essential genes, quasi-essential genes and essential genes in phages includes:
  • Deletable genes with a deletion frequency of less than 5% are determined as quasi-essential genes for phages, and deletable genes with a deletion frequency of more than 5% are determined as non-essential genes for phages.
  • Deletable genes Genes for which no deletions were detected were identified as essential genes for the phage.
  • the frequency of a gene mutation is expressed as a percentage of gene deletion/gene retention in a mutant library.
  • the phage genes can be initially classified into three groups: (1 ) genes deleted in isolated single mutant phage genomes, with a relatively high frequency of gene deletions of more than 5%, were classified as non-essential genes.
  • Other genes whose frequency of gene deletion in the deleteable gene set was ⁇ 5% were classified as quasi-essential genes. Deletion of these genes produces defective phages with a worse growth advantage than mutants that delete non-essential genes.
  • Genes not detected in the deleteable gene set are important for phage growth and classified as essential genes.
  • the minimum phage genome and the phage with stronger bactericidal ability can be obtained by identifying the essential genes, non-essential genes and quasi-essential genes of the phage.
  • eukaryotic viruses can also simplify the genome by deleting genes through high-throughput deletion according to the method provided in the examples of the present application.
  • the phage is correspondingly replaced by eukaryotic virus
  • the host bacterium is correspondingly replaced by host cell
  • the CiPGr plasmid-bacteria is correspondingly replaced by CiPGr plasmid-cell.
  • phage genome simplification method taking tailed phages T7, T4, seszw and selz as examples, the phage genome simplification method, the screening and determination of non-essential genes and quasi-essential genes in phage genes, and the identification of dominant strains will be described in conjunction with specific examples. Form and screen.
  • CiPGr plasmid library design construction and transformation
  • CiPGr sequence libraries of 200 bp were designed for the potential deleteable gene sequences of tailed phages T7, T4, seszw and selz, 2 CiPGr sequence libraries for each phage.
  • the sequence of CiPGr is shown in Figure 2-a, including barcode (20 bp), homology arm (50 ⁇ 2 bp), promoter (36 bp), spacer (20 bp) and primer (24 bp).
  • CiPGr libraries were synthesized on a 6000-strip size DNA chip as shown in Fig. 2-c-1.
  • the cassette-100 (disrupted gene) and cassette-gene cassettes (deleted gene) of the four phages were separated by PCR, and the cycle conditions for PCR separation were: 98°C, 2 minutes; 98°C, 10 s, 58°C , 20 s, 72°C, 6 s; 72°C, 10 min, store at 4°C.
  • the pTarget plasmid target backbone was amplified by PCR, and the plasmid backbone and CiPGr library were purified by gel electrophoresis. Purification was performed after assembling each CiPGr sequence library with the pTarget plasmid backbone by 4 parallel Gibson assemblies to form a CiPGr plasmid library.
  • CiPGr plasmid library into DH5 ⁇ Escherichia coli competent cells, transform each library 2 to 10 times in parallel, and collect the transformed DH5 ⁇ Escherichia coli competent cells; spread the DH5 ⁇ Escherichia coli competent cells on a 15 cm 2 culture dish Add chloramphenicol and kanamycin, and culture overnight at 37°C.
  • CiPGr plasmids were extracted from DH5 ⁇ E. coli transformed cells after the colonies were scraped off the dishes.
  • CiPGr plasmid libraries were transformed into Escherichia coli (Mg1655, containing the spCas9 plasmid) and Salmonella typhimurium (containing the spCas9 plasmid), to obtain the CiPGr plasmid-bacterial library, which was stored at -80°C for future use.
  • PCR was performed on the CiPGr library and sequenced by HiSeq 2500 (Illumina). The low-quality data was removed from the obtained raw sequencing data, and the sequencing data was compared to the designed CiPGr library sequence to determine the 100% identical ratio of each CiPGr library sequence in the plasmid library.
  • CiPGr-containing plasmid-bacterial library stored at -80°C, and after melting on ice, take out 300 ⁇ L of each library, add it to 15 ml of LB medium, add antibiotics and L-arabinose at the same time, and culture overnight at 37°C.
  • Figure 2-c-3 add 1 ml of phage (10 9 PFU/ml), 1 ml of the corresponding host cell containing the plasmid library (10 9 CFU/ml) and 1 ml of LB medium into the shaker tube, and culture at 37°C After 6h, the first generation of mutant phage products were generated.
  • Figure 3 provides a library of phage-deletable genes, showing the frequency of deletion of various genes in the mutant library of phages transferred at the nth transfer.
  • the deletable gene library is produced by transfer of two types of plasmid libraries (gene destruction library, gene deletion library), where the frequency indicates the percentage (log2) of the corresponding reads (deletion or destruction) of the gene in the population, and the corresponding heat Figures were generated by the phatmap R package. "L” indicates deletion of a large segment that we did not design; “P” indicates deletion of one or two bases in the coding region, resulting in premature codon termination.
  • phage genes were monitored by PCR every 10 transfers. 1 ⁇ L of the supernatant was used as a template for PCR using Ex Taq DNA polymerase (Takara, RR01AM), and amplification was performed on the target gene with appropriate primers. Gene deletions were verified by agarose gel electrophoresis and Sanger sequencing. As shown in Figure 4, PCR monitoring of phage gene deletion is provided, and the deletion efficiency of gp4.7 and gp5.3 in the phage T7 genome is detected by PCR and gel electrophoresis. In the figure, the band sizes of gp4.7 and gp5.3 were reduced from 559 bp and 455 bp to 128 bp and 117 bp, respectively.
  • the DNA of the mutant phage library was extracted according to the manual of the phage DNA isolation kit (NORGEN, 46850), and the DNA was sequenced with a HiSeq1500 sequencer. We sequenced the 20th, 30th, 40th, and 50th transfer products of phages T7 and T4, and then every 50 to 100 transfers thereafter.
  • a single mutant phage 10 ⁇ L of overnight cultured host cells, 200 ⁇ L of fresh LB medium were added to a 96-well plate, and a single phage plaque was purified and picked into a 96-well plate. Incubate on a microplate reader at 37°C, and detect OD600 every 10 min for 12 hours; select mutant phages according to the bactericidal curve, that is, the one-step growth curve of phage, and further purify them by drawing the plate method.
  • the method for determining the one-step growth curve of phages is as follows: after the bacteria are streaked, single clones are picked and cultured overnight; the phages are freshly cultured, the concentration is measured, and diluted to 10 5 PFU for later use; 3 tubes, after incubating at 37°C for 2 hours, centrifuge at 7000g at room temperature, remove the supernatant, add 5ml of preheated medium, shake and mix well; add 0.2mmol/L CaCl, add 100 ⁇ L phage, shake and mix well; after standing at room temperature for 5min , add 30ml of preheated culture medium, vortex and mix well; after culturing for a certain period of time, take samples and continue to detect the concentration.
  • Fig. 5 is the phage plaque of mutant phage T7 in the double-layer agar plate
  • Fig. 6 is the bactericidal curve of mutant phage T7 and T4 to MG1655, and the bactericidal curve of mutant phage seszw and selz to Salmonella ST56.
  • T8 mutant phages Mix the mutant phage libraries of different transfers, mix the mutant phage libraries of different transfers (10 5 PFU) and the corresponding logarithmic growth (10 8 PFU) of wild-type host cells Co-cultured for 2 hours to obtain T1 mutant phages; part of the T1 mutant phages were transferred to fresh host cells for culture again, so that a total of 8 transfers were performed, and a total of 16-20 generations were amplified to obtain T8 mutant phages.
  • 10 3 PFU of T8 mutant phage 300 ⁇ L of wild-type host cells and 10 mL of 0.7% LB agar were added to a tube, mixed, poured into LB plates, and incubated overnight at 37°C. Pick 3 different plaques for purification.
  • Fig. 7 is the bactericidal curve of the stronger phage of screening, wherein, a is the bactericidal curve of stronger T7 and T4 mutant strain to MG1655 and stronger seszw and selz mutant strain to Salmonella (ST56); b is T7, seszw, T4 and One-step growth curves of stronger phages from Selz.
  • WT represents the wild-type phage; the data are expressed as the mean ⁇ SD of three experiments; the phage titer was determined by the PFU method.
  • Fig. 8 is the gene map of the stronger phage screened, wherein the black bars indicate gene deletion regions; the black dots indicate point mutations. Arrows indicate genes, and the direction of the arrow corresponds to the direction of transcription and translation.
  • the frequency of a gene mutation is expressed as the percentage of gene deletion/gene retention in a mutant library.
  • CiPGr to four different tailed phages (model phages T7 and T4; Salmonella phages seszw and selz) resulted in mutants of these phages deleting 8%–23% (3.3–35 kbp) of their sequences, yielding simplified Phage backbone.
  • Mutant phages with stronger bactericidal ability than wild-type phages were obtained through screening. We observed that the stronger mutants screened performed faster than their wild-type counterparts in killing the host (5 minutes faster for T7, 2 hours faster for T4, 1 hour faster for seszw, and 2 hours faster for selz).
  • CiPGr is a general and efficient method applicable to novel phages and other eukaryotic viruses without any prior knowledge.

Abstract

一种高通量简化噬菌体基因组骨架的方法，该方法包括如下步骤：设计并合成噬菌体的CiPGr序列文库，将CiPGr序列文库与pTarget质粒骨架组装，形成CiPGr质粒文库；将CiPGr质粒进行转化后提取CiPGr质粒；将CiPGr质粒转化进入含有spCas9质粒的宿主细菌中，得到CiPGr质粒-细菌文库；将野生型噬菌体和对应的CiPGr质粒-细菌文库在培养基中培养，迭代培养，生成突变噬菌体库；对间隔一定转移次数后得到的突变噬菌体库进行测序，确认所述噬菌体中的所有可删除基因。提供的方法，可以实现包括有尾噬菌体在内的所有噬菌体的基因组简化。

Description

高通量简化噬菌体基因组骨架的方法 技术领域
本申请涉及生命科学技术领域，具体涉及一种高通量简化噬菌体基因组骨架的方法。
 背景技术
噬菌体是地球上最丰富、最具基因多样性的生物体。在一个多世纪的时间里，噬菌体研究一直是许多生物发现的关键，它为分子生物学提供了重要的生物技术工具。近年来，病毒宏基因组测序显示，在人类肠道和其他环境中发现了大量噬菌体序列。这些序列的大部分(＞75%)是新型的，超过95%的序列属于有尾双链(ds)DNA噬菌体。此外，噬菌体已被认为是潜在的治疗细菌感染的天然抗菌药物。但同时，噬菌体作为潜在的治疗细菌感染的天然抗菌药物存在的宿主特异性、容易产生抗性等问题，直接影响噬菌体的成药性。因此，科研工作者对噬菌体进行了许多合成生物学方面的努力，以克服这些限制。
无尾噬菌体如M13和X174，具有非常紧凑和较小的基因组(＜10kb)，由于编码有限数量的基因相对简单，使其易于编辑和理解其基因的功能。而有尾噬菌体的基因组通常数量相对较大(14~500 kbp)且呈异常多样化，这使得大规模获得有尾噬菌体简化基因组存在较大挑战性。其中一个挑战是：在基因组规模内，没有有效的方法来识别噬菌体的非必需基因。比如模式噬菌体T7的25个非必需基因，是从最近三四十年的大量研究中积累知识中获得的。噬菌体的扩增完全依赖其宿主，其独特的自我传播特性，使得在细菌中广泛使用的方法（在微生物如大肠杆菌、酵母、芽孢杆菌和支原体中广泛应用的简化基因组方法，如同源重组、Tn5突变等）可能不适合大规模在有尾噬菌体中应用。此外，由于噬菌体基因组的高度多样性的特征，使得通过生物信息方法比对很难获得非必需基因信息。
从头合成简化基因组，需要高通量鉴定噬菌体的非必需基因，但高通量鉴定方法如dCas9方法，有两个原因导致失败：一是由于噬菌体基因组在细菌中会复制很多份，使许多噬菌体基因组的基因表达不能完全被dCas9抑制，导致假阳性结果；第二，噬菌体的基因很多是串联转录的，dCas9抑制上游基因,将导致所有下游基因的抑制表达，导致了假阴性的结果。而Tn5类似的转座子方法同样由于噬菌体独特的生长方式而导致鉴定效率下降。
 技术问题
本申请实施例的目的之一在于：提供一种高通量简化噬菌体基因组骨架的方法。
 技术解决方案
本申请实施例采用的技术方案是：
一种高通量简化噬菌体基因组骨架的方法，包括如下步骤：
设计并合成两种或两种以上噬菌体的多个CiPGr序列文库，将各所述CiPGr序列文库中分别与pTarget质粒骨架组装，形成CiPGr质粒文库；其中，所述CiPGr序列文库包括n个CiPGr序列，n为大于或等于2的自然数，所述CiPGr序列包含gRNA，且各所述CiPGr序列的gRNA不同；
将所述CiPGr质粒文库中的CiPGr质粒进行转化，筛选含有所述CiPGr质粒的转化态细胞，从所述转化态细胞中提取所述CiPGr质粒；将所述CiPGr质粒转化进入含有spCas9质粒的宿主细菌中，得到CiPGr质粒-细菌文库，所述CiPGr质粒-细菌文库中含有n种CiPGr质粒-细菌；
将野生型噬菌体和对应的所述CiPGr质粒-细菌文库在培养基中培养，取突变噬菌体产物转移到新鲜的所述CiPGr质粒-细菌文库中继续培养，重复迭代培养，生成突变噬菌体库；
对间隔一定转移次数后得到的突变噬菌体库进行测序，确认所述噬菌体中的所有可删除基因。
在一个实施例中，所述突变噬菌体库的生成步骤为：
将所述野生型噬菌体和对应的所述CiPGr质粒-细菌文库在培养基中培养，生成第一代突变噬菌体产物，离心所述第一代突变噬菌体产物，取上清液，得到第一代突变噬菌体库；将部分所述第一代突变噬菌体文库和新鲜的所述CiPGr质粒-细菌文库培养基中继续培养，生成第二代突变噬菌体产物，离心所述第二代突变噬菌体产物，取上清液，得到第二代突变噬菌体库；将部分所述第二代突变噬菌体库和新鲜的所述CiPGr质粒-细菌文库在培养基中培养，生成第三代突变噬菌体产物，离心所述第三代突变噬菌体产物，取上清液，得到第三代突变噬菌体库；重复该迭代培养的步骤，使转移次数达到300~600次，收集各突变噬菌体库。
在一个实施例中，所述方法还包括筛选单种具有活性的突变噬菌体。
在一个实施例中，所述筛选单种具有活性的突变噬菌体，包括：
将1×10 3 ~5×10 3  PFU的突变噬菌体库与野生型宿主细胞在固态培养基中培养，随机挑选若干大菌斑和小菌斑进行分离纯化；将过夜培养的宿主细胞和新鲜LB培养基加入到96孔板中，加入单种突变噬菌体后，在酶标仪上培养，每隔一段时间检测一次OD 600，持续12~24小时；根据杀菌曲线选择突变噬菌体，并通过划板法对所选择的所述突变噬菌体进行进一步纯化。
在一个实施例中，所述方法还包括筛选优势突变噬菌体，步骤包括：
把所述突变噬菌体库中的突变噬菌体混合后，与野生型宿主细胞共培养后，转移到新鲜的野生型宿主细胞中再次培养，重复“转移到新鲜的野生型宿主细胞中再次培养”的步骤N-1次，得到TN突变噬菌体；
将1×10 3 ~5×10 3 PFU的TN突变噬菌体、野生型宿主细胞和LB琼脂混合后倒入LB平板，培养过夜；
挑选不同的噬菌斑进行纯化后，将过夜培养的宿主细胞和新鲜LB培养基加入到96孔板中，并将单个斑块提纯后转移到96孔板中，在酶标仪上培养，并且每隔一段时间检测一次OD 600，持续12~24小时；根据杀菌曲线括筛选优势突变噬菌体。
在一个实施例中，所述方法还包括所述噬菌体中非必需基因、准必需基因和必需基因的确定。
在一个实施例中，所述噬菌体中非必需基因、准必需基因和必需基因的确定，包括：
分析所述突变噬菌体文库中突变噬菌体的缺失基因的概率，缺失频率＜5%的可删除基因确定为所述噬菌体的准必需基因，缺失频率＞5%的可删除基因确定为所述噬菌体的非必需基因，未检测到删除的基因确定为所述噬菌体的必需基因。
在一个实施例中，所述CiPGr序列还包含条形码、两条同源臂、启动子和引物。
在一个实施例中，将所述CiPGr质粒文库中的CiPGr质粒转化后，筛选含有所述CiPGr质粒的转化态细胞，从所述转化态细胞中提取所述CiPGr质粒，包括：
将所述CiPGr质粒文库中的CiPGr质粒转化DH5α大肠杆菌感受态细胞，平行转化2~10次，收集转化后的DH5α大肠杆菌感受态细胞；将所述DH5α大肠杆菌感受态细胞铺在含有氯霉素和卡那霉素的培养皿上培养，筛选含有所述CiPGr质粒的DH5α大肠杆菌转化态细胞，从所述DH5α大肠杆菌转化态细胞中提取所述CiPGr质粒。
在一个实施例中，将所述CiPGr序列文库中所有的CiPGr序列分别与pTarget质粒骨架组装的步骤之前，还包括对所述CiPGr序列文库中的所述CiPGr序列按不同噬菌体进行PCR分离。
在一个实施例中，所述PCR分离的循环条件为：
98°C，2分钟；98°C，10 s，58°C，20 s，72°C，6 s；72℃，10分钟，在4°C保存。
在一个实施例中，将所述CiPGr序列文库分别与pTarget质粒骨架组装的方法为：将所述CiPGr序列文库和所述pTarget质粒骨架通过Gibson组装反应进行连接后，纯化处理。
在一个实施例中，将将野生型噬菌体和对应的所述CiPGr质粒-细菌文库在培养基中培养的步骤之前，还包括：将所述CiPGr质粒-细菌文库加入LB培养基中，加入抗生素和L-阿拉伯糖过夜培养。
在一个实施例中，所述重复迭代培养的重复次数为300-600次。
在一个实施例中，所述重复迭代培养的步骤中，定期测定突变噬菌体库的滴定浓度；当得到的突变噬菌体的浓度少于10 5个/ml时，采用野生型宿主细胞培养突变噬菌体库。
在一个实施例中，所述噬菌体为有尾噬菌体、无尾噬菌体或其他真核病毒。
作为一种实施方式，所述噬菌体替代为真核病毒，所述宿主细菌替代为宿主细胞，所述CiPGr质粒-细菌替代为CiPGr质粒-细胞。
 有益效果
本申请实施例提供的高通量简化噬菌体基因组骨架的方法的有益效果在于：针对噬菌体的可删除基因，设计CiPGr序列中gRNA各不相同的CiPGr序列文库，将CiPGr序列与pTarget质粒骨架组装后形成CiPGr质粒（即pTarget质粒），将CiPGr质粒转化进入含有spCas9质粒的宿主细菌，得到CiPGr质粒-细菌文库。此时，宿主细菌中同时含有pTarget质粒和spCas9质粒，CiPGr质粒-细菌文库中不同的单个细菌可能含有不同的pTarget质粒。将野生型噬菌体侵染CiPGr质粒-细菌，pTarget编码编码的向导RNA(gRNA)引导spCas9编码的Cas9核酸酶结合到目标基因上，并使目标基因双链断裂，通过同源序列诱导的同源重组（HR）修复，导致基因缺失或破坏。如果该基因对噬菌体生长不必需，突变噬菌体的后代可以在没有该基因的情况下扩增。通过迭代培养，噬菌体侵染不同的CiPGr质粒-细菌，导致不同的基因被删除，从而不断产生突变噬菌体。突变噬菌体不断转移到新的含有双质粒的细菌即CiPGr质粒-细菌中，可能侵染含有不同pTarget质粒的宿主菌，并删除不同的基因。自上而下的不断高通量删除基因，将使噬菌体基因组上的基因删除不断积累，得到含有不同删除基因的突变噬菌体库，对间隔一定转移次数后得到的突变噬菌体进行测序，直至获取噬菌体基因组骨架。该方法可以快速实现包括有尾噬菌体和无尾噬菌体在内的所有噬菌体的基因组骨架的高通量简化。
 附图说明
为了更清楚地说明本申请实施例中的技术方案，下面将对实施例或示范性技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本申请的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其它的附图。
图1是本申请实施例提供的高通量简化噬菌体基因组骨架的方法流程示意图；
图2是本申请实施例提供的高通量简化噬菌体基因组骨架的方法流程示意图；
图3是本申请实施例1提供的噬菌体可删除基因库；
图4是本申请实施例1提供的突变体中，不同转接次数中噬菌体T7基因组中gp4.7和gp5.3的凝胶电泳图；
图5是本申请实施例1提供的双层琼脂平板中突变噬菌体T7的噬菌斑；
图6是本申请实施例1提供的突变噬菌体T7和T4对MG1655的杀菌曲线；
图7是本申请实施例1提供的比野生型抑菌能力更强噬菌体的杀菌曲线；
图8是本申请实施例1提供的更强突变噬菌体的基因图谱。
 本发明的实施方式
为了使本申请的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本申请进行进一步详细说明。应当理解，此处所描述的具体实施例仅用以解释本申请，并不用于限定本申请。
术语“第一”、“第二”仅用于便于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明技术特征的数量。“多个”的含义是两个或两个以上，除非另有明确具体的限定。
噬菌体合成生物学可以将功能基因或基因线路整合到噬菌体基因组中，增强其在噬菌体治疗及其他不同生物工程应用中的抗菌活性和潜力。这种基因线路整合可能具有挑战性，因为噬菌体颗粒的DNA包装空间有限。噬菌体的附属基因可以帮助噬菌体更好地适应广泛的生态环境，但在给定条件下，它可能是非必要的。这些非必需基因的删除可以在噬菌体基因组中创造一些空间。同时，从噬菌体基因组中去除多余的基因将有助于促进对噬菌体-宿主相互作用模式和噬菌体生理学的理解。这种理解可以进一步促进重新设计更强大的噬菌体基因组，为基本的生物发现铺平道路。
为了解决这些挑战，本申请实施例提供了一种自上而下的不断高通量删除基因以简化基因组的方法，即依赖CRISPR-cas9的迭代简化噬菌体基因组方法（CiPGr）。本申请实施例中，噬菌体可以为有尾噬菌体，也可以为无尾噬菌体。
为了说明本申请所提供的技术方案，以下结合具体附图及实施例进行详细说明。
具体的，参考图1，该方法包括如下步骤：
S10. 设计并合成两种或两种以上噬菌体的多个CiPGr序列文库，将各CiPGr序列文库分别与pTarget质粒骨架组装，形成CiPGr质粒文库。
该步骤中，针对潜在的可删除基因序列，设计噬菌体的CiPGr序列文库，本申请实施例可以同时对两种或两种以上的噬菌体设计CiPGr序列文库，其中，一种噬菌体可以设计一个CiPGr序列文库，也可以设计两个CiPGr序列文库。
各CiPGr序列文库包括n个CiPGr序列，n的取值取决于一种噬菌体中潜在的可删除基因的数量，n为大于或等于2的自然数。本申请实施例中，CiPGr序列至少包括向导RNA（guide RNA，gRNA），且每个CiPGr序列的gRNA各不相同，以匹配一种噬菌体中不同的可删除基因。应当理解的是，噬菌体中已知的必需基因，或者在美国国家生物技术信息中心（NCBI）上噬菌体的同源必需基因，不用设计向导RNA（guide RNA，gRNA）。
在一些实施例中，CiPGr序列包含条形码、两条同源臂、启动子、gRNA和引物。其中，条形码用于区分不同的噬菌体文库，通过条形码引物PCR将不同的噬菌体分开；引物为gRNA序列的一部分，在将“CiPGr序列与pTarget质粒骨架组装”进行Gibson组装时，作为Gibson组装的位点。
本申请实施例将所有CiPGr序列在DNA芯片上合成，实现CiPGr序列文库的批量合成。示例性的，在一个6000条大小的DNA芯片中合成了所有设计的CiPGr序列文库。在一个实施例中，设计CiPGr序列的长度为200bp，以方便基因序列在DNA芯片上的合成，并降低合成成本。示例性的，CiPGr序列为200bp，其中，条形码20 bp，两条同源臂各为50 bp，启动子36 bp，spacer 20 bp，以及引物24 bp。
本申请实施例中，通过PCR技术将多个CiPGr序列文库进行拆分，即对CiPGr序列文库中的CiPGr序列按不同噬菌体进行PCR分离。具体的，通过CiPGr序列上的引物和条形码，对CiPGr序列文库进行PCR分离，将多个CiPGr序列文库拆分成单个CiPGr序列文库。示例性的，PCR分离的循环条件为：98°C，2分钟；98°C，10 s，58°C，20 s，72°C，6 s；72℃，10分钟，在4°C保存。
本申请实施例中，将各CiPGr序列文库分别与pTarget质粒骨架组装，CiPGr序列文库中的CiPGr序列与pTarget质粒骨架组装形成CiPGr质粒，CiPGr序列文库中的CiPGr序列组装后形成的CiPGr质粒，组合形成一个CiPGr质粒文库。
在一些实施例中，pTarget质粒骨架包括Cm R和Ori，其中，Cm R表示氯霉素耐药基因，Ori限位复制的起源。在一些实施例中，pTarget质粒骨架可以通过PCR扩增，PCR扩增得到的pTarget质粒骨架可以采用凝胶电泳法纯化。在一些实施例中，在将CiPGr序列文库与pTarget质粒骨架组装之前，采用凝胶电泳法对CiPGr序列文库进行纯化处理。
在一些实施例中，将各CiPGr序列文库分别与pTarget质粒骨架组装的方法为：将各CiPGr序列文库和pTarget质粒骨架通过Gibson组装反应进行连接后，纯化处理。示例性的，每个CiPGr文库和pTarget质粒骨架的连接都通过4个平行的Gibson组装反应实现。由此，得到含有CiPGr序列的pTarget质粒，且不同的单个宿主细胞，可能含有不同的pTarget质粒。
S20. 将CiPGr质粒文库中的CiPGr质粒进行转化，筛选含有CiPGr质粒的转化态细胞，从转化态细胞中提取CiPGr质粒；将CiPGr质粒转化进入含有spCas9质粒的宿主细菌中，得到CiPGr质粒-细菌文库，CiPGr质粒-细菌文库中含有n种CiPGr质粒-细菌。
该步骤中，将CiPGr质粒文库中的CiPGr质粒进行转化，丰富CiPGr质粒的数量。在一些实施例中，将每个CiPGr质粒文库中的CiPGr质粒转化DH5α大肠杆菌感受态细胞，平行转化2~10次，收集转化后的DH5α大肠杆菌感受态细胞；将DH5α大肠杆菌感受态细胞铺在含有氯霉素和卡那霉素的培养皿上培养，筛选含有CiPGr质粒的DH5α大肠杆菌转化态细胞，从DH5α大肠杆菌转化态细胞中提取CiPGr质粒。
示例性的，将DH5α大肠杆菌感受态细胞铺在含有氯霉素和卡那霉素的培养皿上培养的方式，可以为：37℃条件下培养过夜。
将CiPGr质粒转化进入含有spCas9质粒的宿主细菌中，得到CiPGr质粒-细菌文库，所述CiPGr质粒-细菌文库中含有n种CiPGr质粒-细菌。此时，宿主细菌中包含双质粒系统即pTarget质粒和spCas9质粒。其中，含有spCas9质粒的宿主细菌根据噬菌体的种类确定。示例性的，当噬菌体含有有尾噬菌体T7、T4、seszw和selz中的至少一种时，含有spCas9质粒的宿主细菌可以为含有spCas9质粒的大肠杆菌(Mg1655)和含有spCas9质粒的鼠伤寒沙门氏菌。
本申请实施例可以将得到的CiPGr质粒-细菌文库在-80℃的条件下保存。
在一些实施例中，为了评估CiPGr质粒-细菌文库中设计的CiPGr序列文库的覆盖情况，进行针对CiPGr序列文库的PCR，并通过HiSeq 2500(Illumina)测序。将得到的原始测序数据去除低质量数据后，将测序数据比对到设计的CiPGr文库序列，并确定每个CiPGr文库序列在质粒库中100%一致的比例。
S30. 将野生型噬菌体和对应的CiPGr质粒-细菌文库在培养基中培养，取突变噬菌体产物转移到新鲜的CiPGr质粒-细菌文库中继续培养，重复迭代培养，生成突变噬菌体库。
该步骤中，取CiPGr质粒-细菌文库，加入培养基中，培养过夜。当CiPGr质粒-细菌文库为-80℃的条件下保存的CiPGr质粒-细菌文库时，将CiPGr质粒-细菌文库冰上融化后，取CiPGr质粒-细菌文库加入培养基。示例性的，培养温度课为37℃；示例性的，培养基可为LB培养基。在一些实施例中，为了防止其他菌对CiPGr质粒-细菌文库的污染，在培养基中加入抗生素和L-阿拉伯糖。
在一个实施例中，将CiPGr质粒-细菌加入LB培养基中，加入抗生素和L-阿拉伯糖过夜培养。
将野生型噬菌体和对应的CiPGr质粒-细菌文库在培养基中培养，噬菌体侵染对应的CiPGr质粒-细菌文库，将噬菌体DNA注进对应的CiPGr质粒-细菌文库的细胞内。而CiPGr质粒-细菌中pTarget编码的gRNA引导spCas9编码的Cas9核酸酶结合到野生型噬菌体上的目标基因上，并使目标基因双链断裂。通过同源序列诱导的同源重组(HR)修复，导致噬菌体基因缺失或破坏。如果该基因对噬菌体生长不必需，突变噬菌体的后代可以在没有该基因的情况下扩增。野生型噬菌体侵染CiPGr质粒-细菌文库中不同的CiPGr质粒-细菌，导致不同的基因被删除，从而产生突变噬菌体产物。本申请实施例中，野生型噬菌体和对应的CiPGr质粒-细菌文库在培养基中进行第一次培养后产生的突变噬菌体产物纯化，形成第一代突变噬菌体库。
应当理解的是，本申请实施例中，对应的CiPGr质粒-细菌文库，是指野生型噬菌体能够侵染的CiPGr质粒-细菌。
取突变噬菌体产物转移到新鲜的CiPGr质粒-细菌文库中继续培养，使突变噬菌体产物转移到新鲜的含有双质粒的细菌中，可能侵染含有不同CiPGr质粒-细菌，并删除不同的基因。经过重复迭代培养，将突变噬菌体产物不断转移到新的含有双质粒的细菌即CiPGr质粒-细菌中，将使野生型噬菌体基因组上的基因删除不断积累。同时，具有生长优势的突变株，可以产生更多的子代，并最终在突变种群中占据主导地位，成为优势株。在一些实施例中，重复迭代培养的重复次数为300-600次，从而有利于简便地检测出非必需基因、准必需基因和必需基因。
应当理解的是，本申请实施例所指的新鲜的CiPGr质粒-细菌文库，是指没有与野生型噬菌体共培养的CiPGr质粒-细菌文库，并不一定是新鲜配置的CiPGr质粒-细菌文库。但应该理解的是，对于同一个野生型噬菌体而言，重复迭代培养的CiPGr质粒-细菌文库、重复迭代培养的CiPGr质粒-细菌文库以及第一次培养过程中的CiPGr质粒-细菌文库的类型一致。
在一些实施例中，突变噬菌体库的生成步骤为：
将野生型噬菌体和对应的CiPGr质粒-细菌文库在培养基中培养，生成第一代突变噬菌体产物，离心第一代突变噬菌体产物，取上清液，得到第一代突变噬菌体库；将部分第一代突变噬菌体库和新鲜的CiPGr质粒-细菌文库培养基中继续培养，生成第二代突变噬菌体产物，离心第二代突变噬菌体产物，取上清液，得到第二代突变噬菌体库；将部分第二代突变噬菌体库和新鲜的CiPGr质粒-细菌文库在培养基中培养，生成第三代突变噬菌体产物，离心第三代突变噬菌体产物，取上清液，得到第三代突变噬菌体库；重复该迭代培养的步骤，使转移次数达到300~600次，收集各突变噬菌体库。
在这种情况下，突变噬菌体产物不断转移到新的含有双质粒的细菌即CiPGr质粒-细菌中，使野生型噬菌体基因组上的基因删除不断积累，从而形成缺失不同基因的突变噬菌体，各种不同基因缺失的突变噬菌体形成突变噬菌体库。突变噬菌体库中各种不同缺失基因形成可删除基因集。
上述方法中，每一次将培养后得到的突变噬菌体库的一部分，加入新鲜的CiPGr质粒-细菌文库，另一部分在低温条件下储存，已进行进一步的分析。在一些实施例中，重复迭代培养的步骤中，定期测定突变噬菌体库的滴定浓度；当得到的突变噬菌体的浓度少于10 5个/ml时，采用野生型宿主细胞培养突变噬菌体库。由于传代导致突变噬菌体数量下降，在这种情况下，可以通过野生型宿主细胞培养扩大突变噬菌体的数量或菌体浓度。示例性的，每10次转接,测定一次突变噬菌体库的滴定浓度。
S40. 对间隔一定转移次数后得到的突变噬菌体库进行测序，确认所述噬菌体中的所有可删除基因。
该步骤中，对间隔一定转移次数后得到的突变噬菌体库进行测序，获取突变噬菌体的可删除基因信息。该步骤可以在得到突变噬菌体库后，对储存的所有突变噬菌体进行测序，也可以在培养突变噬菌体的过程中，对得到的突变噬菌体进行测序。
在一些实施例中，采用PCR监测一次噬菌体基因的删除。示例性的，采用一μL的突变噬菌体上清液作为PCR的模板，使用Ex Taq DNA聚合酶(Takara,RR01AM)，通过适当的引物在目标基因上进行扩增，通过琼脂糖凝胶电泳和Sanger测序验证基因的删除。
本申请实施例中，为了监测基因缺失的效率，按照噬菌体DNA分离试剂盒(NORGEN,46850)的手册，提取突变噬菌体库的DNA，采用HiSeq1500测序仪进行DNA测序。示例性的，对噬菌体T7和T4的第20、30、40和50次转接产物进行测序，然后每50到100次转接进行一次测序。
在一种可能的实施方式中，本申请实施例提供的方法还可以筛选单种具有活性的突变噬菌体。在一些实施例中，筛选单种具有活性的突变噬菌体，包括：
将1×10 3 ~5×10 3 PFU的突变噬菌体库与野生型宿主细胞在固态培养基中培养，随机挑选若干大菌斑和小菌斑进行分离纯化；将过夜培养的宿主细胞和新鲜LB培养基加入到96孔板中，加入单种突变噬菌体后，在酶标仪上培养，每隔一段时间检测一次OD 600，持续12~24小时；根据杀菌曲线选择突变噬菌体，并通过划板法对所选择的所述突变噬菌体进行进一步纯化。
示例性的，以有尾噬菌体T7、T4、seszw和selz为例，每一种噬菌体两个文库（共形成8个CiPGr质粒-细菌文库）。为了获得单种突变噬菌体，将1×10 3 PFU的突变噬菌体库与300 μL的野生型宿主细胞和10 mL浓度为0.7%的LB琼脂被加入到一个管中混合，并倒入平板，并在37℃条件下过夜培养。随机挑取8个大的和8个小的斑块，然后进行分离纯化。为了确定单个突变噬菌体的杀菌效果，将10 μL过夜培养的宿主细胞、200 μL新鲜LB培养基加入到96孔板中，并将单个噬菌斑纯化并挑取到96孔板中，在酶标仪上于37℃条件下进行培养，并且每10 min检测一次OD600，持续12小时；根据杀菌曲线即噬菌体一步生长曲线选择突变噬菌体，并通过划板法进行进一步纯化，得到单种具有活性的突变噬菌体。
示例性的，噬菌体一步生长曲线测定方法为：菌划线后，挑单克隆过夜培养；噬菌体新鲜培养，测浓度，稀释到10 5 PFU备用；将培养基预热后，1%体积接种10ml，共3管，37℃培养2h后，室温7000g离心，去上清，加入5ml预热的培养基，震荡混匀；加入0.2mmol/L的CaCl，加入100μL噬菌体，震荡混匀；室温静置5min后，加入预热的培养基30ml，震荡混匀；培养一定时间后取样持续检测浓度。
本申请实施例通过上述步骤，可以快速、高通量地获得具有活性的简化噬菌体基因组，特别是对于有大量未知功能基因的新噬菌体。
将野生型噬菌体和对应的所述CiPGr质粒-细菌文库进行迭代培养的过程中，野生型噬菌体基因组上的基因删除不断积累，同时，具有生长优势的噬菌体突变体，可以产生更多的子代，并最终在突变种群中占据主导地位，成为优势株。
本申请实施例中，分离噬菌体突变体并进行单克隆测序，可以得到简化的噬菌体基因组；而把噬菌体突变文库混合在一起竞争培养，可以分离出具有比野生型噬菌体更强的突变株。在一种可能的实施方式中，本申请实施例提供的方法还可以筛选优势突变噬菌体，筛选步骤包括：
把突变噬菌体库中的突变噬菌体混合后，与对应的野生型宿主细胞共培养后，转移到新鲜的野生型宿主细胞中再次培养，重复“转移到新鲜的野生型宿主细胞中再次培养”的步骤N-1次，得到TN突变噬菌体（(其中，T表示转接，N表示转移数)）；
将1×10 3 ~5×10 3 PFU的TN突变噬菌体、野生型宿主细胞和LB琼脂混合后倒入LB平板，培养过夜；
挑选不同的噬菌斑进行纯化后，将过夜培养的宿主细胞和新鲜LB培养基加入到96孔板中，并将单个斑块提纯后转移到96孔板中，在酶标仪上培养，并且每隔一段时间检测一次OD 600，持续12~24小时；根据杀菌曲线括筛选优势突变噬菌体。
示例性的，以有尾噬菌体T7、T4、seszw和selz为例，把不同转接的突变噬菌体库混合在一起，混合的突变噬菌体库（10 5 PFU）与相应的对数生长（10 8 PFU）的野生型宿主细胞共培养2小时，得到T1突变噬菌体；将部分T1突变噬菌体再次转移到新鲜宿主细胞培养，这样一共进行8次转移，共扩增16-20代，得到T8突变噬菌体。将10 3 PFU的T8突变噬菌体、300μL野生型宿主细胞和10 mL浓度为0.7%的LB琼脂被加入到一个管中混合，并倒入LB平板，在37℃条件下培养过夜。挑取3个不同的噬菌斑进行纯化。为了确定单中突变噬菌体的杀菌效果，将10 μL过夜培养的宿主细胞、200 μL新鲜LB培养基加入到96孔板中，并将单个斑块提纯并转移到96孔板中，在酶标仪上，37℃进行培养，并且每10 min检测一次OD600，持续12小时。
本申请实施例可以通过对突变噬菌体库进行宏基因组测序，简便地检测出非必需基因、准必需基因和必需基因。在一种可能的实施方式中，本申请实施例提供的方法还包括所述噬菌体中非必需基因、准必需基因和必需基因的确定。
在一些实施例中，噬菌体中非必需基因、准必需基因和必需基因的确定，包括：
分析突变噬菌体文库中突变噬菌体的缺失基因的概率，缺失频率＜5%的可删除基因确定为噬菌体的准必需基因，缺失频率＞5%的可删除基因确定为噬菌体的非必需基因，可删除基因未检测到删除的基因确定为噬菌体的必需基因。
应当理解的是，一个基因突变的频率表示为在一个突变文库中基因删除/基因保留所占的百分比。将野生型噬菌体和对应的所述CiPGr质粒-细菌文库进行迭代培养的过程中，野生型噬菌体基因组上的基因删除不断积累，根据迭代培养的结果，可以初步将噬菌体基因分类为三组：(1)在分离的单个突变噬菌体基因组中缺失的基因，基因缺失的频率相对较高，超过5%，被归类为非必需基因。(2)可删除基因集中基因缺失的频率< 5%的其他基因被归类为准必需基因。这些基因的缺失会产生有缺陷的噬菌体，其增长优势比删除非必需基因的突变体更差。(3)在可删除基因集中未检测到的基因对噬菌体的生长是重要的，归类为必需基因。
示例性的，以有尾噬菌体T7、T4、seszw和selz为例，在分离的单个的突变噬菌体基因组中，检测到的基因缺失的多数(T7 100%、T4 92.4%、seszw 94.1%、selz 98.4%)在转接的突变噬菌体文库中，显示了相对较高的频率＞5%,这表明这些基因对噬菌体生长的重要性低于其他基因（频率＜5%）。
本申请实施例可以通过鉴定噬菌体的必需基因、非必需基因和准必需基因，获得最小噬菌体基因组和杀菌能力更强噬菌体。
应当理解的是，真核病毒也可以参照本申请实施例提供的方法，通过高通量删除基因以简化基因组。上述方法中，噬菌体对应替代为真核病毒，宿主细菌对应替代为宿主细胞，CiPGr质粒-细菌对应替代为CiPGr质粒-细胞。
下面参考图2，以有尾噬菌体T7、T4、seszw和selz为例，结合具体实施例进行说明噬菌体基因组简化方法，以及噬菌体基因中非必需基因、准必须基因的筛选和确定，以及优势株的形成和筛选。
（1）CiPGr质粒库设计、构建和转化
针对有尾噬菌体T7、T4、seszw和selz的潜在的可删除基因序列，设计200 bp的CiPGr序列文库共8个，每种噬菌体2个CiPGr序列文库。其中，CiPGr序列如图2-a所示，包含条形码(20 bp)、同源臂(50×2 bp)、启动子(36 bp)、spacer(20 bp)和引物(24 bp)。
如图2-c-1所示，在6000条大小的DNA芯片上合成所有设计的CiPGr文库。4种噬菌体的cassette-100（破坏基因）和cassette-gene cassettes（删除基因）通过PCR分离，其中，PCR分离的循环条件为：98°C，2分钟；98°C，10 s，58°C，20 s，72°C，6 s；72℃，10分钟，在4°C保存。
如图2-c-2所示，利用PCR扩增pTarget质粒目标骨架，用凝胶电泳法纯化了质粒骨架和CiPGr文库。通过4个平行的Gibson组装将每个CiPGr序列文库与pTarget质粒骨架组装形成CiPGr质粒文库后，进行纯化。将纯化后的CiPGr质粒文库转化DH5α大肠杆菌感受态细胞，每个文库平行转化2~10次，收集转化后的DH5α大肠杆菌感受态细胞；将DH5α大肠杆菌感受态细胞铺在15 cm 2培养皿中，加入氯霉素和卡那霉素，37℃培养过夜。将菌落从培养皿上刮下来后，从DH5α大肠杆菌转化态细胞中提取CiPGr质粒。将8个CiPGr质粒库转化进入大肠杆菌(Mg1655，含有spCas9质粒)和鼠伤寒沙门氏菌(含有spCas9质粒)中，得到CiPGr质粒-细菌文库，保存在-80℃备用。
该步骤中，为了评估质粒库中设计的CiPGr文库的覆盖情况，进行针对CiPGr文库的PCR，并通过HiSeq 2500(Illumina)测序。将得到的原始测序数据去除低质量数据，将测序数据比对到设计的CiPGr文库序列，确定了每个CiPGr文库序列在质粒库中100%一致的比例。
（2）突变噬菌体库的生成和宏基因组测序
取出-80℃保存的含有CiPGr质粒-细菌文库，冰上融化后，每个文库取出300 μL，加入15 ml的LB培养基中，同时加入抗生素和L-阿拉伯糖，37℃过夜培养。如图2-c-3，1 ml噬菌体(10 9 PFU/ml)、1 ml相应的含有质粒文库宿主细胞(10 9 CFU/ml)和1 ml LB培养基加入摇菌管中，37℃培养6h后，生成第一代突变噬菌体产物。取上述的1 mL第一代突变噬菌体产物，离心取上清，再次转移到1 ml新鲜的含有CiPGr质粒-细菌文库和1ml LB培养基中培养；如上述过程，转接进行了300-600次。图3提供了噬菌体可删除基因库，表示在第n次转接的噬菌体突变库的各种基因删除频率。该可删除基因库由两种类型的质粒库(基因破坏文库，基因删除文库)转接产生，其中，频率表示该基因相应reads(缺失或破坏)在群体中的百分比(log2)，相应的热图由phatmap R软件包生成。“L”表示删除了一个我们没有设计的大片段；“P”表示编码区域的一个或两个碱基缺失，导致密码子过早终止。
其中，在每一次转接中产生的上清，1ml用于传代，1ml在4℃中储存，以进行进一步的分析。每10次转接，测定一次突变噬菌体库的滴定浓度。如果突变噬菌体浓度少于10 5个/ml，野生型的宿主细胞被用来培养突变的噬菌体库。
每10次转接用PCR监测一次噬菌体基因的删除。1 μL的上清被用作PCR的模板，使用Ex Taq DNA聚合酶(Takara,RR01AM)，并通过适当的引物在目标基因上进行扩增。通过琼脂糖凝胶电泳和Sanger测序验证基因的删除。如图4，提供了PCR监测噬菌体基因删除情况，通过PCR和凝胶电泳检测噬菌体T7基因组中gp4.7和gp5.3的删除效率。图中，gp4.7和gp5.3的条带大小分别从559 bp和455 bp减少到128 bp和117 bp。
为了监测基因缺失的效率，按照噬菌体DNA分离试剂盒(NORGEN,46850)的手册，提取突变噬菌体库的DNA，DNA用了HiSeq1500测序仪进行测序。我们对噬菌体T7和T4的第20、30、40和50次转接产物进行测序，然后每50到100次转接进行一次测序。
（3）筛选、表征和基因组测序单个分离的突变噬菌体
如图2-c-4，为了获得单个的突变噬菌体，将10 3 PFU的突变噬菌体库与300 μL的野生型宿主细胞和10 mL的浓度为0.7%的LB琼脂被加入到一个管中混合，并倒入平板，在37℃过夜培养。随机挑取8个大的和8个小的斑块，然后进行分离纯化。
为了确定单个突变噬菌体的杀菌效果，将10 μL过夜培养的宿主细胞，200 μL新鲜LB培养基加入到96孔板中，并将单个噬菌斑纯化并挑取到96孔板中。在酶标仪上，37℃进行培养，并且每10 min检测一次OD600，持续12小时；根据杀菌曲线即噬菌体一步生长曲线选择突变噬菌体，并通过划板法进行进一步纯化。其中，噬菌体一步生长曲线测定的方法为：菌划线后，挑单克隆过夜培养；噬菌体新鲜培养，测浓度，稀释到10 5 PFU备用；将培养基预热后，1%体积接种10ml，共3管，37℃培养2h后，室温7000g离心，去上清，加入5ml预热的培养基，震荡混匀；加入0.2mmol/L的CaCl，加入100μL噬菌体，震荡混匀；室温静置5min后，加入预热的培养基30ml，震荡混匀；培养一定时间后取样持续检测浓度。
图5为双层琼脂平板中突变噬菌体T7的噬菌斑；图6为突变噬菌体T7和T4对MG1655的杀菌曲线，以及突变噬菌体seszw和selz对沙门氏菌ST56的杀菌曲线。
（4）筛选、表征和基因组测序更强突变噬菌体
把不同转接的突变噬菌体库混合在一起，把不同转接的突变噬菌体库混合在一起，混合的突变噬菌体库（10 5 PFU）与相应的对数生长（10 8 PFU）的野生型宿主细胞共培养2小时，得到T1突变噬菌体；将部分T1突变噬菌体再次转移到新鲜宿主细胞培养，这样一共进行8次转移，共扩增16-20代，得到T8突变噬菌体。将10 3 PFU的T8突变噬菌体、300μL野生型宿主细胞和10 mL浓度为0.7%的LB琼脂被加入到一个管中混合，并倒入LB平板，在37℃条件下培养过夜。挑取3个不同的噬菌斑进行纯化。为了确定单中突变噬菌体的杀菌效果，将10 μL过夜培养的宿主细胞、200 μL新鲜LB培养基加入到96孔板中，并将单个斑块提纯并转移到96孔板中，在酶标仪上，37℃进行培养，并且每10 min检测一次OD600，持续12小时。
图7是筛选的更强噬菌体的杀菌曲线，其中，a为更强T7和T4突变株对MG1655和更强seszw和selz突变株对沙门氏菌 (ST56)的杀菌曲线；b为T7、seszw、T4和selz的更强噬菌体的一步生长曲线。图中，WT表示野生型噬菌体；数据以三个实验的平均值±SD表示；采用PFU法测定噬菌体滴度。
图8是筛选的更强噬菌体的基因图谱，其中，黑色条块表示基因缺失区域；黑色的点表示点突变。箭头表示基因，箭头的方向对应转录和翻译的方向。
（5）数据分析
一个基因突变的频率表示为在一个突变文库中基因删除/基因保留所占的百分比。我们使用了开源的软件breseq(版本0.28.0)来预测每一个突变文库的点突变和基因删除；突变噬菌体的基因组组拼接使用SOAP-denove (V2.04-r241)软件。
CiPGr应用于四种不同的有尾噬菌体(模式噬菌体T7和T4；沙门氏菌噬菌噬菌体seszw和selz)，导致这些噬菌体的突变体删除8%~23%(3.3-35 kbp)的序列，产生了简化噬菌体骨架。突变噬菌体库的宏集测序表明，非必需基因和准必需基因占这些噬菌体基因总数的46.7%~65.4%。准必需基因(24%~26%)的丧失可能对噬菌体扩增造成严重损害，使得相应的突变体在突变文库中逐渐消失，导致在分离的单个突变体的基因组中检测不到准必需基因的删除。
通过筛选获得了比野生型噬菌体杀菌能力更强的突变噬菌体。我们观察到，在杀死宿主方面，所筛选的更强突变体的表现比其野生型噬菌体更快（T7快5分钟，T4快2小时，seszw快1小时，selz快2小时）。
上述结果表明，CiPGr是一种通用的、有效的方法，适用于没有任何先验知识的新型噬菌体和其他真核病毒。
以上仅为本申请的可选实施例而已，并不用于限制本申请。对于本领域的技术人员来说，本申请可以有各种更改和变化。凡在本申请的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本申请的权利要求范围之内。

Claims (16)

  1. 高通量简化噬菌体基因组骨架的方法，其特征在于，包括如下步骤：
    设计并合成两种或两种以上噬菌体的多个CiPGr序列文库，将各所述CiPGr序列文库中分别与pTarget质粒骨架组装，形成CiPGr质粒文库；其中，所述CiPGr序列文库包括n个CiPGr序列，n为大于或等于2的自然数，所述CiPGr序列包含gRNA，且各所述CiPGr序列的gRNA不同；
    将所述CiPGr质粒文库中的CiPGr质粒进行转化，筛选含有所述CiPGr质粒的转化态细胞，从所述转化态细胞中提取所述CiPGr质粒；将所述CiPGr质粒转化进入含有spCas9质粒的宿主细菌中，得到CiPGr质粒-细菌文库，所述CiPGr质粒-细菌文库中含有n种CiPGr质粒-细菌；
    将野生型噬菌体和对应的所述CiPGr质粒-细菌文库在培养基中培养，取突变噬菌体产物转移到新鲜的所述CiPGr质粒-细菌文库中继续培养，重复迭代培养，生成突变噬菌体库；
    对间隔一定转移次数后得到的突变噬菌体库进行测序，确认所述噬菌体中的所有可删除基因。
  2. 根据权利要求1所述的高通量简化噬菌体基因组骨架的方法，其特征在于，所述突变噬菌体库的生成步骤为：
    将所述野生型噬菌体和对应的所述CiPGr质粒-细菌文库在培养基中培养，生成第一代突变噬菌体产物，离心所述第一代突变噬菌体产物，取上清液，得到第一代突变噬菌体库；将部分所述第一代突变噬菌体文库和新鲜的所述CiPGr质粒-细菌文库培养基中继续培养，生成第二代突变噬菌体产物，离心所述第二代突变噬菌体产物，取上清液，得到第二代突变噬菌体库；将部分所述第二代突变噬菌体库和新鲜的所述CiPGr质粒-细菌文库在培养基中培养，生成第三代突变噬菌体产物，离心所述第三代突变噬菌体产物，取上清液，得到第三代突变噬菌体库；重复该迭代培养的步骤，使转移次数达到300~600次，收集各突变噬菌体库。
  3. 根据权利要求1所述的高通量简化噬菌体基因组骨架的方法，其特征在于，所述方法还包括筛选单种具有活性的突变噬菌体。
  4. 根据权利要求3所述的高通量简化噬菌体基因组骨架的方法，其特征在于，所述筛选单种具有活性的突变噬菌体，包括：
    将1×10 3 ~5×10 3  PFU的突变噬菌体库与野生型宿主细胞在固态培养基中培养，随机挑选若干大菌斑和小菌斑进行分离纯化；将过夜培养的宿主细胞和新鲜LB培养基加入到96孔板中，加入单种突变噬菌体后，在酶标仪上培养，每隔一段时间检测一次OD 600，持续12~24小时；根据杀菌曲线选择突变噬菌体，并通过划板法对所选择的所述突变噬菌体进行进一步纯化。
  5. 根据权利要求1至4任一项所述的高通量简化噬菌体基因组骨架的方法，其特征在于，所述方法还包括筛选优势突变噬菌体，步骤包括：
    把所述突变噬菌体库中的突变噬菌体混合后，与对应的野生型宿主细胞共培养后，转移到新鲜的野生型宿主细胞中再次培养，重复“转移到新鲜的野生型宿主细胞中再次培养”的步骤N-1次，得到TN突变噬菌体；
    将1×10 3 ~5×10 3 PFU的TN突变噬菌体、野生型宿主细胞和LB琼脂混合后倒入LB平板，培养过夜；
    挑选不同的噬菌斑进行纯化后，将过夜培养的宿主细胞和新鲜LB培养基加入到96孔板中，并将单个斑块提纯后转移到96孔板中，在酶标仪上培养，并且每隔一段时间检测一次OD 600，持续12~24小时；根据杀菌曲线括筛选优势突变噬菌体。
  6. 根据权利要求1至4任一项所述的高通量简化噬菌体基因组骨架的方法，其特征在于，所述方法还包括所述噬菌体中非必需基因、准必需基因和必需基因的确定。
  7. 根据权利要求6所述的高通量简化噬菌体基因组骨架的方法，其特征在于，所述噬菌体中非必需基因、准必需基因和必需基因的确定，包括：
    分析所述突变噬菌体文库中突变噬菌体的缺失基因的概率，缺失频率＜5%的可删除基因确定为所述噬菌体的准必需基因，缺失频率＞5%的可删除基因确定为所述噬菌体的非必需基因，可删除基因未检测到删除的基因确定为所述噬菌体的必需基因。
  8. 根据权利要求1至4任一项所述的高通量简化噬菌体基因组骨架的方法，其特征在于，所述CiPGr序列还包含条形码、两条同源臂、启动子和引物。
  9. 根据权利要求1至4任一项所述的高通量简化噬菌体基因组骨架的方法，其特征在于，将所述CiPGr质粒文库中的CiPGr质粒转化后，筛选含有所述CiPGr质粒的转化态细胞，从所述转化态细胞中提取所述CiPGr质粒，包括：
    将所述CiPGr质粒文库中的CiPGr质粒转化DH5α大肠杆菌感受态细胞，平行转化2~10次，收集转化后的DH5α大肠杆菌感受态细胞；将所述DH5α大肠杆菌感受态细胞铺在含有氯霉素和卡那霉素的培养皿上培养，筛选含有所述CiPGr质粒的DH5α大肠杆菌转化态细胞，从所述DH5α大肠杆菌转化态细胞中提取所述CiPGr质粒。
  10. 根据权利要求1至4任一项所述的高通量简化噬菌体基因组骨架的方法，其特征在于，将各所述CiPGr序列文库分别与pTarget质粒骨架组装的步骤之前，还包括对所述CiPGr序列文库中的所述CiPGr序列按不同噬菌体进行PCR分离。
  11. 根据权利要求10所述的高通量简化噬菌体基因组骨架的方法，其特征在于，所述PCR分离的循环条件为：
    98°C，2分钟；98°C，10 s，58°C，20 s，72°C，6 s；72℃，10分钟，在4°C保存。
  12. 根据权利要求1至4任一项所述的高通量简化噬菌体基因组骨架的方法，其特征在于，将所述CiPGr序列文库分别与pTarget质粒骨架组装的方法为：将所述CiPGr序列文库和所述pTarget质粒骨架通过Gibson组装反应进行连接后，纯化处理。
  13. 根据权利要求1至4任一项所述的高通量简化噬菌体基因组骨架的方法，其特征在于，将野生型噬菌体和对应的所述CiPGr质粒-细菌文库在培养基中培养的步骤之前，还包括：将所述CiPGr质粒-细菌文库加入LB培养基中，加入抗生素和L-阿拉伯糖过夜培养。
  14. 根据权利要求1至4任一项所述的高通量简化噬菌体基因组骨架的方法，其特征在于，所述重复迭代培养的步骤中，定期测定突变噬菌体库的滴定浓度；当得到的突变噬菌体的浓度少于10 5个/ml时，采用野生型宿主细胞培养突变噬菌体库。
  15. 根据权利要求1至4任一项所述的高通量简化噬菌体基因组骨架的方法，其特征在于，所述噬菌体为有尾噬菌体或无尾噬菌体。
  16. 根据权利要求1至15任一项所述的高通量简化噬菌体基因组骨架的方法，其特征在于，所述噬菌体替代为真核病毒，所述宿主细菌替代为宿主细胞，所述CiPGr质粒-细菌替代为CiPGr质粒-细胞。
PCT/CN2022/079314 2022-03-04 2022-03-04 高通量简化噬菌体基因组骨架的方法 WO2023164934A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2022/079314 WO2023164934A1 (zh) 2022-03-04 2022-03-04 高通量简化噬菌体基因组骨架的方法

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2022/079314 WO2023164934A1 (zh) 2022-03-04 2022-03-04 高通量简化噬菌体基因组骨架的方法

Publications (1)

Publication Number Publication Date
WO2023164934A1 true WO2023164934A1 (zh) 2023-09-07

Family

ID=87882693

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/079314 WO2023164934A1 (zh) 2022-03-04 2022-03-04 高通量简化噬菌体基因组骨架的方法

Country Status (1)

Country Link
WO (1) WO2023164934A1 (zh)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170283779A1 (en) * 2016-03-28 2017-10-05 The Charles Stark Draper Laboratory, Inc. Bacteriophage engineering methods
US20190330643A1 (en) * 2018-04-25 2019-10-31 The Catholic University Of America Engineering of bacteriophages by genome editing using the crispr-cas9 system

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170283779A1 (en) * 2016-03-28 2017-10-05 The Charles Stark Draper Laboratory, Inc. Bacteriophage engineering methods
US20190330643A1 (en) * 2018-04-25 2019-10-31 The Catholic University Of America Engineering of bacteriophages by genome editing using the crispr-cas9 system

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
HATOUM-ASLAN, A.: "Phage Genetic Engineering Using CRISPR–Cas Systems", VIRUSES, vol. 10, 19 June 2018 (2018-06-19), XP055877011, DOI: 10.3390/v10060335 *
SHENGJIAN YUAN, MA YINGFEI: "Advances and applications of phage synthetic biology", SYNTHETIC BIOLOGY JOURNAL, vol. 1, no. 6, 15 December 2020 (2020-12-15), pages 635 - 655, XP093087599, DOI: 10.12211/2096-8280.2020-027 *
TAO, PAN ET AL.: "Engineering of Bacteriophage T4 Genome Using CRISPR-Cas9", ACS SYNTHETIC BIOLOGY, vol. 6, no. 10, 20 October 2017 (2017-10-20), XP055943284, DOI: 10.1021/acssynbio.7b00179 *
YUAN SHENGJIAN, SHI JUAN, JIANG JIANRONG, MA YINGFEI: "Genome-scale top-down strategy to generate viable genome-reduced phages", NUCLEIC ACIDS RESEARCH, OXFORD UNIVERSITY PRESS, GB, vol. 50, no. 22, 9 December 2022 (2022-12-09), GB , pages 13183 - 13197, XP093087598, ISSN: 0305-1048, DOI: 10.1093/nar/gkac1168 *

Similar Documents

Publication Publication Date Title
CN107083392B (zh) 一种CRISPR/Cpf1基因编辑系统及其在分枝杆菌中的应用
Thomason et al. Recombineering: genetic engineering in bacteria using homologous recombination
Tao et al. Unexpected evolutionary benefit to phages imparted by bacterial CRISPR-Cas9
CN106995813B (zh) 基因组大片段直接克隆和dna多分子组装新技术
US11377645B2 (en) Compositions and methods using Cas9 with enhanced spacer acquisition function
Toussaint et al. Transposable phages, DNA reorganization and transfer
Cronan Improved plasmid-based system for fully regulated off-to-on gene expression in Escherichia coli: Application to production of toxic proteins
Zhang et al. Biological characteristics and genomic analysis of a Stenotrophomonas maltophilia phage vB_SmaS_BUCT548
Chouikha et al. Insights into the infective properties of YpfΦ, the Yersinia pestis filamentous phage
Xue et al. High frequency of a novel filamentous phage, VCYϕ, within an environmental Vibrio cholerae population
JP7436493B2 (ja) 次世代シーケンスにおいて低サンプルインプットを扱うための正規化対照
JP2008504832A (ja) バクテリオファージにおける組換え遺伝子の産生
Yuan et al. Genome-scale top-down strategy to generate viable genome-reduced phages
Du et al. Composition and function of viruses in sauce-flavor baijiu fermentation
EP4189084A1 (en) Methods of making unbiased phage libraries
Faber et al. Saturation mutagenesis genome engineering of infective φx174 bacteriophage via unamplified oligo pools and golden gate assembly
US9315803B2 (en) Method for the preparation of modified bacteriophages by insertion of random sequences in the targeting proteins of said bacteriophages
WO2023164934A1 (zh) 高通量简化噬菌体基因组骨架的方法
Zhang et al. Replication protein Rep provides selective advantage to viruses in the presence of CRISPR-Cas immunity
CN114934059B (zh) 高通量简化噬菌体基因组骨架的方法
CN116162616A (zh) 高效诱变方法在提高噬菌体辅助进化效率中的应用
CN113583931B (zh) 魏氏柠檬酸杆菌ansB基因敲除突变株及其应用
CN114657213A (zh) 一种猪急性腹泻综合征冠状病毒人工染色体重组载体及其构建方法和应用
Zhang et al. Unexplored diversity and ecological functions of transposable phages
Billaud et al. The Clostridium-infecting filamentous phage CAK1 genome analysis allows to define a new potential clade of Tubulavirales

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22929377

Country of ref document: EP

Kind code of ref document: A1