WO2025078633A1 - Method for cell-free bacteriophage synthesis - Google Patents

Abstract

The invention relates to a cell-free method for synthesizing bacteriophages, comprising: (a) in vitro assembling overlapping DNA fragments into a phage genome, in a ligase-free assembling reaction, comprising: (a1) digesting the overlapping DNA fragments by an exonuclease, and (a2) annealing the overlapping DNA fragments to assemble a phage genome, and (b) cell-free transcribing and translating the phage genome. The invention also relates to phages synthesized by the method and to their applications.

Description

METHOD FOR CELL-FREE BACTERIOPHAGE SYNTHESIS

FIELD OF THE INVENTION

[0001] This disclosure pertains to the field of cell-free bacteriophage synthesis.

BACKGROUND

Bacteriophages (phages) comprise an immense reservoir of biotechnologically-relevant bioactive materials such as DNA engineering tools (Salmond & Fineran) (e.g., CRISPR technologies (Stanley & Maxwell)) with broad applications in phage therapy (Dedrick et al, Harada et al, Lemire et al, Endersen et al, Matsuzaki et al), nanotechnology (Daube et al, Vonshak et al), and vaccine scaffolds engineering (Bundy et al, Lu et al). Despite their potential and abundance, there are engineering limitations to phages’ widespread use (Principi et al, Pryor et al, Kiro et al, Liu et al, Grigonyte et al).

[0002] Cell-free transcription-translation (TXTL) offers an alternative experimental setting to achieve phage engineering and synthesis, complementary to and potentially faster than existing methods (Rustad et al, Garenne et al, 2021 ). TXTL provides an unparalleled speed to design, build and test DNAs (Garenne et al, 2019). TXTL has proven effective for prototyping gene circuits (Noireaux et al, 2003, Takahashi et al, Sun et al, Tayar et al), manufacturing biologies (Pardee et al), or building biological systems from the ground up (Noireaux et al, 2004, Godino et al, Gaut et al). As it gains in strength and versatility (Shin & Noireaux, Garamella, 2016, Garenne et al, 2019), TXTL can be challenged for executing larger DNAs. In this regard, the cell-free synthesis (CFS) of infectious T7 and T4 phages in an E. coli X.T\_ system demonstrates that natural genomic DNAs encoding for tens of genes can be achieved in vitro (Shin et al, 2012, Rustad et al, 2017, Rustad et al, 2018). The CFS of phages reduces the usage of bacteria and pathogens and provides a boundary-free and accessible environment meant for bioengineering innovation.

[0003] However, current phage engineering using TXTL lack a rapid and scalable method to assemble, synthesize and select phage genomes. Existing procedures are either time-consuming and relatively costly, or are achieved in buffers that do not interface well with TXTL, which requires additional purification steps after assembly.

[0004] Accordingly, there is a need for a low-cost and more efficient method for cell-free synthesis of bacteriophages.

Summary

[0005] The present invention provides a method for synthesizing bacteriophages.

[0006] One aspect of the invention relates to a cell-free method for synthesizing bacteriophages, comprising:

(a) in vitro assembling overlapping DNA fragments into a phage genome, in a ligase-free assembling reaction, comprising: (a1 ) digesting the overlapping DNA fragments by an exonuclease, and

(a2) annealing the overlapping DNA fragments to assemble a phage genome; and

(b) cell-free transcribing and translating the phage genome.

[0007] In one embodiment, the method comprises a step of inactivating the exonuclease after step (a1 ) and before step (a2), preferably wherein the inactivation is by heat treatment.

[0008] In one embodiment, step (a) is preceded by a step of amplifying a template phage genome by PCR to produce the overlapping DNA fragments.

[0009] In one embodiment, the overlapping DNA fragments comprise overlapping DNA sequences of at least 5 bp at their 3’- and 5’-ends.

[0010] In one embodiment, the overlapping DNA fragments comprise orthogonal overlapping sequences at their 3’- and 5’-ends such that two adjacent DNA fragments specifically anneal together.

[0011] In one embodiment, the exonuclease is a 3’->5’ exonuclease, preferably is exonuclease III.

[0012] In one embodiment, the exonuclease is the sole enzyme used in step (a).

[0013] In one embodiment, the phage genome is not purified between step (a) and (b).

[0014] In one embodiment, the overlapping DNA fragments comprise one or more genomic insertions, deletions and/or mutations with respect to the template phage genome.

[0015] In one embodiment, the overlapping DNA fragments comprises one or more heterologous nucleotide sequences, in particular one or more heterologous genes.

[0016] In one embodiment, step (b) comprises contacting the phage genome with an E coli lysate, preferably comprising the core RNA polymerase and sigma factor 70.

[0017] In one embodiment, the phage is selected from T7, T6 , T5, lambda, VPAE1 , and FelixOl .

[0018] In one embodiment, more than 10⁹’ PFU/ml, in particular more than 10¹⁰ PFU/ml phages are produced.

[0019] In one embodiment, the synthetized phage contains 2 assembly errors or less per DNA fragment junction, preferably 1 assembly error or less or no assembly error per junction.

DETAILED DESCRIPTION OF THE INVENTION

Sequence listing

SEQ ID NO:1 shows the sequence of the p70a-deGFP plasmid.

SEQ ID NO:2 shows the sequence of the T7p14-deGFP plasmid.

SEQ ID NO: 3 shows the sequence of the p70a-T7RNAP plasmid.

SEQ ID NO: 4 shows the sequence of the T7p14-Mcherry plasmid. SEQ ID NO: 5 shows the sequence of the T7-gp10 plasmid.

SEQ ID NO: 6 shows the sequence of primer T7A-s.

SEQ ID NO: 7 shows the sequence of primer T7A-as.

SEQ ID NO: 8 shows the sequence of primer T7B-s.

SEQ ID NO: 9 shows the sequence of primer T7B-as.

SEQ ID NO: 10 shows the sequence of primer T7C-S.

SEQ ID NO: 11 shows the sequence of primer T7C-as.

SEQ ID NO: 12 shows the sequence of primer T7D-s.

SEQ ID NO: 13 shows the sequence of primer T7D-as.

SEQ ID NO: 14 shows the sequence of primer T7D2-as.

SEQ ID NO: 15 shows the sequence of primer T7E-s.

SEQ ID NO: 16 shows the sequence of primer T7E-as.

SEQ ID NO: 17 shows the sequence of primer T7F-s1 .

SEQ ID NO: 18 shows the sequence of primer gp1 -oho1 -as.

SEQ ID NO: 19 shows the sequence of primer gp1 -oho2-s.

SEQ ID NO: 20 shows the sequence of primer gp10-oho1 -as.

SEQ ID NO: 21 shows the sequence of primer gp10-oho2-s.

SEQ ID NO: 22 shows the sequence of primer gp17-oho1-as.

SEQ ID NO: 23 shows the sequence of primer gp17-oho2-s.

SEQ ID NO: 24 shows the sequence of primer oho1 -mcherry-s.

SEQ ID NO: 25 shows the sequence of primer oho2-mcherry-as.

SEQ ID NO: 26 shows the sequence of primer mcherry-tt-s.

SEQ ID NO: 27 shows the sequence of primer mcherry-tt-as.

SEQ ID NO: 28 shows the sequence of primer T7C-s3.

SEQ ID NO: 29 shows the sequence of primer T7C-as3.

SEQ ID NO: 30 shows the sequence of primer T7mini-A-s.

SEQ ID NO: 31 shows the sequence of primer T7mini-A-24-as.

SEQ ID NO: 32 shows the sequence of primer T7mini-B-24-s.

SEQ ID NO: 33 shows the sequence of primer T7mini-B-21-as.

SEQ ID NO: 34 shows the sequence of primer T7mini-C-21 -s. SEQ ID NO: 35 shows the sequence of primer T7mini-C-23-as.

SEQ ID NO: 36 shows the sequence of primer T7mini-D-23-s.

SEQ ID NO: 37 shows the sequence of primer T7mini-D-as.

SEQ ID NO: 38 shows the sequence of primer T7mini-E-s.

SEQ ID NO: 39 shows the sequence of primer T7mini-E-as.

SEQ ID NO: 40 shows the sequence of primer T7mini-F-s.

SEQ ID NO: 41 shows the sequence of primer T7mini-F-as.

SEQ ID NO: 42 shows the sequence of primer backbone-s.

SEQ ID NO: 43 shows the sequence of primer backbone-as.

SEQ ID NO: 44 shows the sequence of primer gp10-s.

SEQ ID NO: 45 shows the sequence of primer gp10-as.

SEQ ID NO: 46 shows the amino acid sequence of exonuclease III.

Definitions

[0020] In order that the present disclosure be more readily understood, certain terms are defined. Additional definitions are set forth throughout the detailed description.

[0021] As used herein, the term “bacteriophage” or “phage” refers to a duplodnaviria virus that infects and replicates within bacteria and archaea. Bacteriophages are composed of proteins that encapsulate a DNA or RNA genome. Their genomes may encode as few as four genes (e.g. MS2) and as many as hundreds of genes. Phages replicate within the bacterium following the injection of their genome into its cytoplasm.

[0022] As used herein, the term “exonuclease” refers to an enzyme that cleaves nucleotides one at a time from the end of a polynucleotide chain via a hydrolyzing reaction that breaks phosphodiester bonds at either the 3’ or 5’ end. The “exonuclease” can be a 3’ to 5’ exonuclease or a 5’ to 3' exonuclease. E. coli exonuclease I and exonuclease III are two commonly used 3’-exonucleases that have 3’-exonucleolytic single-strand degradation activity. E. coli exonuclease VII and T7- exonuclease Gene 6 are two commonly used 5’->3’ exonucleases that have 5’-exonucleolytic singlestrand degradation activity.

[0023] As used herein the term, “ligase” refers to an enzyme that establishes a phosphodiester bond between adjacent nucleotides in a nucleic acid.

[0024] As used herein, the term “polymerase” refers to an enzyme which catalyzes the polymerization of ribonucleoside triphosphates (including deoxyribonucleoside triphosphates) to make nucleic acid chains. [0025] As used herein, the term “cell-free”, e.g. in “cell-free method” or “cell-free reaction”, refers to a method or reaction, such as a biosynthetic reaction (e.g., transcription reaction, translation reaction, or both) which is carried out in vitro in the absence of living cells. A cell-free reaction is supported by a set of reagents capable of providing for or supporting such a biosynthetic reaction. For example, to provide for a transcription reaction, a cell-free reaction may comprise promoter-containing DNA, RNA polymerase, ribonucleotides, and a buffer system. Cell-free systems can be prepared using enzymes, coenzymes, and other subcellular components either isolated or purified from eukaryotic or prokaryotic cells, including recombinant cells, or prepared as extracts or fractions of such cells. A cell-free system can be derived from a variety of sources, including, but not limited to, eukaryotic and prokaryotic cells, such as bacteria including, but not limited to, E. coli, thermophilic bacteria and the like.

[0026] As used herein, the term "in vitro" refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments can consist of, but are not limited to, test tubes. The term "in vivo" refers to the natural environment (e.g., an animal or a cell) and to processes or reaction that occur within a natural environment.

[0027] As used herein, the term “synthesizing” e.g. in “synthesizing a phage genome” encompasses any technique that results in the production of a phage genome. In particular, it encompasses assembling, preferably in vitro assembling, a phage genome from nucleic acid fragments, e.g. DNA fragments.

[0028] As used herein, the terms “polypeptide” “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acids (natural or unnatural, e.g. synthetic) of any length. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component.

[0029] As used herein, the term “anneal” refers to specific interactions between strands of nucleotides wherein the strands bind to one another substantially based on complementarity between the strands as determined by Watson-Crick base pairing or Hoogstein-type hydrogen bonding. Base-stacking and hydrophobic interactions may also contribute to duplex stability. Conditions for hybridizing probes and primers to complementary and substantially complementary target sequences are well known. In general, whether such annealing takes place is influenced by, among other things, the length of the polynucleotides and the complementarity between the bases, the pH, the temperature, the presence of mono- and divalent cations, the proportion of G and C nucleotides in the hybridizing region, the viscosity of the medium, and the presence of denaturants. Such variables influence the time required for hybridization. Thus, the preferred annealing conditions will depend upon the particular application. Such conditions, however, may be routinely determined by the person of ordinary skill in the art without undue experimentation. It will be appreciated that complementarity need not be perfect; there can be a small number of base pair mismatches that will minimally interfere with hybridization between the target sequence and the single-stranded nucleic acids of the present teachings. However, if the number of base pair mismatches is so great that no hybridization can occur under minimally stringent conditions, then the sequence is generally not a complementary target sequence. Thus, “complementarity” herein is meant that the probes or primers are sufficiently complementary to the target sequence to hybridize under the selected reaction conditions to achieve the ends of the present teachings.

[0030] Percent complementarity of an antisense nucleic acid compound with a region of a target nucleic acid can be determined by alignment. For example, an antisense compound in which 18 out of 20 nucleotides are complementary to a target sequence, i.e. with two mismatches between its sequence and the target sequence, would represent 90% complementarity. Percent complementarity of an antisense compound with a region of a target nucleic acid can also be determined using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) pileup program, or any of sequence comparison algorithms such as BLAST, FASTA or CLUSTALW.

[0031] As used herein, the term “identity” refers to the sequence similarity between two polypeptide molecules or between two nucleic acid molecules. When a position in both compared sequences is occupied by the same base or same amino acid residue, then the respective molecules are identical at that position. The percentage of identity between two sequences corresponds to the number of matching positions shared by the two sequences divided by the number of positions compared and multiplied by 100. Generally, a comparison is made when two sequences are aligned to give maximum identity (optimal alignment). The identity may be calculated by alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) pileup program, or any of sequence comparison algorithms such as BLAST, FASTA or CLUSTALW.

[0032] As used herein, the term "heterologous " refers to a nucleic acid or polypeptide sequence that is not in its natural environment. For example, a heterologous sequence includes a sequence from one species introduced into another species, e.g. introduced from a foreign species in a bacteriophage genome. A heterologous sequence also includes a sequence native to an organism that has been altered in some way (e.g., mutated, added in multiple copies, linked to nonnative regulatory sequences, etc). Heterologous nucleic acids are distinguished from endogenous nucleic acids in that the heterologous nucleic acid sequences are typically joined to DNA sequences that are not found naturally associated with the nucleic acid sequence in the genome or are associated with portions of the genome not found in nature (e.g., genes expressed in loci where the gene is not normally expressed).

[0033] As used herein, the term "recombinant" refers to a non-natural DNA, protein, cell, seed, or organism that is the result of genetic engineering and was created by human intervention. A "recombinant DNA molecule" is a DNA molecule that does not naturally occur and as such is the result of human intervention, such as a DNA molecule comprised of a combination of at least two DNA sequences heterologous to each other.

[0034] The present disclosure provides a cell-free method to achieve phage genome engineering, synthesis, and selection. The method couples a cell-free DNA assembly procedure with cell-free transcription and translation to synthesize phages in a rapid way, e.g. T7 phages in under one day. The method enables basic biology probing of phage packaging and phage-host interactions and can be used iteratively to rapidly perform permissive phage engineering and selection, without the need of cellular amplification or other synthetic means for genotype-phenotype coupling, such as cell-sized emulsion droplets.

[0035] The method of the invention provides many advantages over existing phage synthesis methods:

(i) it enables gene addition, deletion, mutation and permutations concurrently at any position in a single DNA assembly reaction,

(ii) it provides cell-free DNA assembly,

(iii) it provides a DNA assembly method which interfaces seamlessly with cell-free transcription and translation, with no need for intermediate steps, e.g. purification.

(iv) it is low cost; and

(v) it is easy to handle and technically accessible from start to end.

[0036] The method described herein thus provides a rapid, technically accessible, and low-cost method for phage engineering. Its DNA assembly efficiency surpasses in vivo existing methods and other cell-free phage engineering reports based on Gibson assembly and additional purification, which result in low phage cell-free synthesis (< 10⁵ PFU/ml). This efficiency is mirrored by the ease of engineering deletions and gene insertions, demonstrating the versatility and accessibility of this workflow.

[0037] In one aspect, the invention relates to a cell-free method for synthesizing bacteriophages, comprising:

(a) in vitro assembling overlapping DNA fragments into a phage genome, comprising

(a1 ) contacting the overlapping DNA fragments by an exonuclease, and

(a2) annealing the digested overlapping DNA fragments to assemble a phage genome, and

(b) cell-free transcribing and translating the phage genome.

[0038] The DNA fragments used in step (a) typically are double stranded DNA polynucleotides. Such fragments may be obtained by PCR amplification, e.g. from a template phage genome. Other means to obtain DNA fragments are also encompassed by the invention, such as synthetic DNA obtained from chemical synthesis.

[0039] Once assembled, the DNA fragments form a phage genome. The DNA fragments therefore comprise one or more phage genome sequences. In addition to the phage sequence, the DNA fragments can also contain heterologous sequences, e.g. heterologous genes or chimeric genes.

[0040] The number of DNA fragments and their size are tailored depending on the size of the phage genome to assemble as well as of the size of recombinant fragments to be introduced. The inventors have shown that long fragments can be used to reconstruct phage genomes. In some embodiments, the DNA fragments are from 100 bp to 25 kbp, in particular from 100 bp to 20 kbp, more particularly from 100 bp to 15 kbp. Small fragments can be used. For example, small recombinant fragments of less than 5kbp can be used to introduce a heterologous gene to be introduced within a phage genome. In some embodiments, 2 or more fragments are assembled into a phage genome. In some embodiments, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more or 10 or more fragments are assembled into a phage genome. In some embodiments, the DNA fragments comprise one or more recombinant fragments.

[0041] By “overlapping DNA fragments” it is meant that two adjacent DNA fragments comprise overlapping sequences at their end, wherein said overlapping sequences are complementary such that, after digestion by exonuclease, two adjacent DNA fragments can anneal with each other. In other terms, the sense strand of a DNA fragment comprises a sequence complementary to the antisense strand of an adjacent DNA fragment. Two fragments are said adjacent is they are intended to be joined next to each other in the assembled phage genome.

[0042] The overlapping sequences have a sufficient length to allow annealing, in particular ligase- free annealing of the fragments. Overlapping sequences of at least 5 bp are preferred, in particular at least 10 bp, more preferably at least 15 bp, still more preferably at least 20 bp. Typically, overlapping sequences of from 10 bp, 15 bp, 20 bp, 25 bp, 30 bp, 35 bp, 40 bp, 45 bp, 50 bp, 55 bp, 60 bp, 65 bp, 70 bp, 75 bp, 80 bp, 85 bp, 90 bp, 95 bp or 100 bp can be used. In particular, overlapping sequences of from 5 to 100 bp can be used, in particular from 5 to 75 bp, from 5 to 50 bp, from 10 to 100 bp, from 10 to 75 bp, from 10 to 50 bp, from 20 to 100 bp, from 20 to 75 bp or from 20 to 50 bp.

[0043] In some aspects of the invention, the overlapping sequences are orthogonal oligonucleotide sequences. The orthogonal oligonucleotide sequences can be heterologous sequences added at the end of DNA fragments. The orthogonal sequences are unique for each pair of adjacent DNA fragments, such that one end of a DNA fragment only anneals to one end of an adjacent DNA fragment and not to a non-adjacent DNA fragment. Using a library of orthogonal oligonucleotides as overlapping sequences allow to assemble the phage genome in the desired order. This embodiment is particularly useful to insert recombinant fragments of DNA at a desired position within the genome. It prevents re-assembly of the DNA fragments into a non-edited phage, i.e. phage which reassemble without the recombinant DNA fragment. In some embodiments, the method can comprise a prior step of synthesizing DNA fragments with orthogonal oligonucleotide sequences, wherein two orthogonal oligonucleotide sequences of two adjacent DNA fragments are overlapping. The orthogonal oligonucleotide sequences are added, e.g., by PCR amplification. The orthogonal overlapping sequences can have at least 5 bp, in particular at least 10 bp, preferably at least 20 pb.

[0044] The assembly of all DNA fragments typically forms a linear or circular DNA molecule which reconstructs a phage genome. Said genome can be genome-engineered, or can be unengineered, i.e. identical or substantially identical to a template phage genome, e.g. a naturally-occuring phage genome. [0045] The assembly of the DNA fragments is preferably carried out as a ligase-free reaction. By “ligase-free reaction”, it is meant herein that the step of assembling the overlapping DNA fragments is carried out in the absence of ligase in the reaction mix. Alternatively or in addition, the annealing of the DNA fragments is preferably carried out in the absence of polymerase in the reaction mix. Alternatively or in addition, the assembly is carried out in the absence of IIS endonucleases in the reaction mix. The inventors have shown that seamless genome assembly could be obtained without any ligase activity and without any polymerase activity in the reaction mix. Preferably, the exonuclease is the sole enzyme used in the in vitro assembly of the DNA fragments. In some embodiments, the exonuclease has no polymerase activity. Alternatively or in addition, the assembly is carried out in the absence of IIS endonucleases in the reaction mix.

[0046] According to the invention, the DNA fragments are contacted with an exonuclease at step (a1 ). The outer ends of the DNA fragments are digested by the exonuclease, containing the overlapping sequences, thereby generating protruding overhangs which can anneal.

[0047] The exonuclease is preferably a 3’->5’ exonuclease. Particularly preferred exonucleases include exonuclease III, exonucleases which are homologous to exonuclease III and exonucleases having a comparable activity to exonuclease III. Exonuclease III is an enzyme that catalyzes the stepwise removal of mononucleotides from 3'-hydroxyl termini of duplex DNA. A limited number of nucleotides are removed during each binding event, resulting in coordinated progressive deletions within the population of DNA molecules. The preferred substrates are blunt or recessed 3'-termini, although the enzyme also acts at nicks in duplex DNA to produce single-strand gaps. 3'-protruding termini are resistant to cleavage. Exonuclease III has also been reported to have RNase H, 3'- phosphatase and AP-endonuclease activities. As used herein, exonuclease III includes E. coli exonuclease III (reference sequence Uniprot P09030 as set forth in SEQ ID NO:46). Homologous enzymes are also encompassed by the present invention. The invention encompasses wild-type exonuclease III as well as variant exonucleases III that maintain 3’->5’ exonuclease activity. Preferably, the exonuclease has at least 40%, preferably at least 50%, still preferably at least 60% or 70%, more preferably at least 80 or 90%, most preferably at least 95% or 99% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO:46. Preferably, the exonuclease has at least 10% of the 3’->5’ activity of exonuclease III more preferably at least 20%, even more preferably at least 30%, 40% or 50%, still preferably at least 60%, 70% or 80%, most preferably at least 90%, 95% or 99% of the 3’->5’ activity of exonuclease III. Testing exonuclease activity may be carried out by measuring the fluorescence level of fluorescence probes, e.g. using the 3’ to 5’ Exonuclease Activity Assay (Sigma-Aldrich, #MAK416) Exonuclease, e.g. with a reference exonuclease III from New England Biolabs (#M0206S). Exonuclease III is commercially-available from various sources, for instance from New England Biolabs (#M0206S). In some embodiments, the exonuclease has 3'-phosphatase activity. In some embodiments, the exonuclease has AP- endonuclease activity. In some embodiments, the exonuclease has RNase H activity.

[0048] Preferably, the exonuclease has at least 40% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO:46, and has 3’->5’ exonuclease activity. In particular, %, the exonuclease has at least 50%, more particularly at least 60% or 70%, still more particularly at least 80 or 90%, most particularly at least 95% or 99% amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO:46, and has 3’->5’ exonuclease activity.

[0049] Preferably, the exonuclease has at least 10% of the 3’->5’ activity of exonuclease III. More preferably the exonuclease has at least 20%, even more preferably at least 30%, 40% or 50%, still preferably at least 60%, 70% or 80%, most preferably at least 90%, 95% or 99% of the 3’->5’ activity of exonuclease III, the sequence of which is set forth in SEQ ID NO:46.

[0050] Contacting the exonuclease with the DNA fragments is preferably carried out at a temperature of less than 20°C, preferably less than 15°C. Preferably, the reaction mix containing the DNA fragments is put on ice before being contacted with the exonuclease. Low temperatures of contacting allow to control exonuclease activity.

[0051] The method advantageously comprises a step of inactivating the exonuclease after contacting the DNA fragments with the exonuclease and before annealing the DNA fragments. Inactivation enables to stop the exonuclease activity after digestion of the ends of the DNA fragments, such as to make annealing of the fragments possible. Preferably, a heat treatment is applied to inactivate the exonuclease. The reaction medium may be heated at a temperature of 60°C or more, preferably 70 °C or more, for instance a temperature of about 75°C.The heat treatment is applied for at least 30s, e.g. from 30s to 10mn. Heat treatment may be applied immediately after contacting the DNA fragments with the exonuclease. Indeed, exonuclease activity during the period of temperature increase to the inactivation temperature is generally sufficient to produce digested fragments which are capable of annealing and assemble a phage genome. Digestion time may also depend on the size of the annealing fragments.

[0052] Annealing of the digested ends is preferably carried out at a temperature of less than 60°C, preferably less than 50°C, more preferably less than 45°C, in particular from 20 to 45°C, preferably from 20 to 40°C or from 20 to 35°C. Annealing is advantageously carried out at room temperature. The digested DNA fragments may be annealed for at least 1 minutes, preferably at least 2 minutes. In particular, the digested DNA fragments may be annealed from 1 to 20 minutes minutes., preferably 1 to 10 minutes, still preferably 2 to 10 minutes, most preferably about 3 to 10 minutes. Annealing is carried out without addition of a ligase and/or polymerase, contrary to the Gibson assembly method. Moreover, annealing can be effected in the same reaction mix as the exonuclease digestion, contrary to other existing methods which require an annealing buffer. Hence, no purification step is required between steps (a1 ) and (a2), i.e. exonuclease digestion, and annealing.

[0053] The inventors have shown that the method of the invention reliably assembles the phage genomes, i.e. it generates minimal or no assembly errors at the interface of the assembled DNA fragments. The assembly errors can be checked by sequence alignments with the DNA fragments used before assembly and/or with a templage phage genome. Preferably, the assembled genome has more than 95% sequence identity, preferably more than 98% sequence identity, still preferably more than 99% sequence identity, most preferably about 100% sequence identity with respect to each of the DNA fragments and/or with respect to a template phage genome. Preferably, the assembled genome has 5 nucleotide changes or less, preferably 3 nucleotide changes or less, still preferably 2 nucleotide changes or less, even more preferably 1 nucleotide changes or less, most preferably no nucleotide changes at each interface of the assembled DNA fragments. Sequence comparison with a template phage genome excludes the changes which were voluntarily introduced in the synthetic phage genome (e.g. intended gene mutations, insertions, deletions or permutations).

[0054] In step (b), the assembled phage genomes are submitted to a cell-free transcription and translation step. According to the invention, both the transcription and the translation are carried out cell-free.

[0055] Various cell-free transcription and translation systems can be used, for instance the hybrid T7 cell-free platform that couples the T7 bacteriophage transcription to the translation of an organism such as E. coli. Another cell-free transcription and translation system which can be used is the PURE system, a fully purified cell-free TXTL, also based on the T7 transcription machinery.

[0056] Preferably, cell-free transcription and translation can use an E. coli cell-free system that is independent on the bacteriophage transcription machinery and uses the endogenous E. coil RNA polymerase and housekeeping sigma factor 70. Typically, such TXTL reaction medium is composed of an E. coli lysate, salts, and buffers that provide both the 20 amino acids and an energy mixture such as an ATP regeneration system. The lysate contains the translation machinery (e.g., E. coli core RNA polymerase, RNAP, and sigma factor 70) and the translation components (e.g., ribosomes and tRNA). The translation speed in a cell-free reaction is typically of between 5 and 10 nucleotides per second. To initiate transcription, RNAP and sigma factor 70 (o70) form a holoenzyme to bind to the promoter, in this case P70a, specifically recognized by o70. Because transcription is based on RNAP-o70 from E. coli, the TX repertoire is composed of hundreds of regulatory elements present in E. coli and other bacteria. The other six E. coli sigma factors are not present in the lysate, and therefore must be expressed and used with their specific promoters. Translation is carried out at a rate of roughly two amino acids per second by the same ribosomal machinery present in E. coli.

[0057] In some embodiments, cell-free transcription and translation comprises adding an E. coli lysate to the assembled phage genome. In some embodiments, the E. coli lysate is a lysate from E. coli strain BL21 -ArecBCD Rosetta2. Preferably, the E. coli lysate comprises the E. coli core RNA polymerase and sigma factor 70. In some aspects, the method comprises further adding an energy mixture for ATP regeneration, wherein said energy mixture comprise a phosphate donor and a carbon source. An example of reaction mix for a TXTL reaction comprises comprised E. coli cell lysate, an energy and amino acid mixtures, maltodextrin (30 mM) and ribose (30 mM), magnesium (2-5 mM) and potassium (50-100 mM), PEG8000 (3-4%), water and the DNA to be expressed. The reaction can be incubated at 25-35°C, e.g. around 29 °C,

[0058] Preferably, the reaction mix containing the assembled genome is not purified between steps (a) and (b). An advantage that may be provided by the method of the invention is that the reactions in step (a) are compatible with the performance of subsequent steps of cell-free transcription and translation, without purification. Accordingly, in step (b), the cell-free transcription and reagents, typically including the E. coli lysate, can be added directly to the reaction mixture of step (a), comprising the assembled phage genome.

[0059] The method of the invention can be used to assemble and express various types of phages. . In some embodiments, the phage is a filamentous phage. In some embodiments, the phage is an obligate lytic phage. In some embodiments, the phage is a non-lytic phage. In some embodiments, the phage is selected from the T7, T6, T5, lambda, VPAE160, and FelixOl phages. In some embodiments, the phage is a phage that contains nonessential regions that allow exogenous gene insertions.

[0060] In some embodiment, the synthesized phage comprises at least 10kbp, in particular at least 20 kbp or 30 kbp, more particularly at least 50 kbp, still more particularly at least 75 kbp, even more particularly at least 100 kbp in its genome, most particularly at least 200kbp in its genome.

[0061] In some aspects of the invention, the synthetic phage genome comprises one or more mutations, deletions, insertions or permutations in comparison to a template phage, e.g. a naturally occurring template phage. Genes which can be advantageously mutated include genes which control the infectivity of a phage towards its host.

[0062] Such mutation can be introduced within the DNA fragments, e.g. by PCR amplification using primers comprising the mutation.

[0063] In some aspects of the invention, the synthetic phage genome comprises one or more deleted endogenous genes in comparison to a template phage, e.g. a naturally occurring template phage. The deleted endogenous gene is preferably an early gene (class I) or a DNA metabolism and replication (class II) gene. Preferably, the deleted gene is selected from genes 1 .1 , 1 .2, 1 .3, 1 .4, 1 .5, 1 .6, 3.8, 4.3, 4.5 and 4.7 of the T7 phage or orthologous genes thereof in other phages.

[0064] The inventors have generated a T7-mini phage, demonstrating that the phage genome is permissible to deletions of large portions of the genome, i.e more than 4 kbp. Preferably, the phage genome assembled in the method of the invention comprises 1 kbp or more, preferably 2 kbp or more, still preferably 3 kbp or more, most preferably 4 kbp or more deleted endogenous nucleotide sequences in its genome in comparison to a template phage, e.g. a naturally occurring template phage. In some embodiments, the phage assembled in the method of the invention comprises at least 1 %, preferably at least 2%, still preferably at least 5%, even more preferably at least 7% or 10% deleted endogenous nucleotide sequences in comparison to a template phage, e.g. a naturally occurring template phage. Accordingly, in one aspect, the invention relates to a synthetic phage, in particular, a T7 phage, comprising 1 kbp or more, preferably 2 kbp or more, still preferably 3 kbp or more, most preferably 4 kbp or more deleted endogenous nucleotide sequences in comparison to a template phage, e.g. a naturally occurring template phage. The invention also relates to a synthetic phage, in particular a T7 phage, comprising at least 1 %, preferably at least 2%, still preferably at least 5%, even more preferably at least 7% or 10% deleted endogenous sequences in comparison to a template phage, e.g. a naturally occurring template phage. [0065] The inventors have also relieved the T7 capsid volume limit of 2kbp on new gene insertions, through the use of a reduced phage genome such as the T7-mini phage. Preferably, the phage genome assembled in the method of the invention comprises more than 2 kbp, preferably more than 4 kbp, still preferably more than 5 kbp of inserted heterologous nucleotide sequences in its genome in comparison to a template phage, e.g. a naturally occurring template phage. In one aspect, the invention also relates to a synthetic phage, in particular a T7 phage, comprising more than 2 kbp, preferably more than 4 kbp, still preferably more than 5 kbp of heterologous nucleotide sequences in comparison to a template phage, e.g. a naturally occurring template phage. In some embodiments, the invention relates to a synthetic phage comprising at least 5%, preferably at least 10%, still preferably at least 15% or 20%, even more preferably at least 25% or 30%, most preferably at least 40% or 50% heterologous sequences in its genome.

[0066] Various heterologous genes in the phage genome can be introduced in the phage genome with the method of the invention. In some embodiments, the heterologous gene is a fusion protein, in particular a fusion protein of an endogenous phage protein and an exogenous polypeptide. Preferred fusion proteins are fusion proteins of a phage coat protein and a heterologous polypeptide, allowing applications such as phage display. The heterologous polypeptide may be a peptide from a phage peptide library.

[0067] The method also relates to a phage synthesized according to the method disclosed herein. Any aspect of the method as described in the present disclosure is applicable to the phage. In particular, the phage can be genome-engineered, i.e. comprise one or more mutations, deletions, insertions or permutations with respect to a template phage genome.

[0068] The synthetic phage assembled according to the method can be used in various applications for which phages, in particular engineered phages, are used in biology and medicine. This includes phage display, phage therapy, in particular for human and animal treatment, enzyme discovery, gene cluster discovery, synthesis of protein nanostructures or use as a vector to deliver therapeutic genes to a target cell.

[0069] The invention also relates to a kit comprising :

(i) a bacteriophage, a genome of a bacteriophage, or overlapping DNA fragments, wherein said fragments are designed to assemble into a phage genome upon treatment by an exonuclease;

(ii) an exonuclease or a polynucleotide encoding an exonuclease;

(iii) at least one reagent for cell-free transcription and translation.

[0070] Preferably, said kit does not comprise a ligase or a polynucleotide encoding a ligase.

[0071] Preferably, said reagent for cell-free transcription and translation is a cell or a cell lysate, in particular a bacterial cell or cell lysate, in particular an E. co// cell or E. co// lysate.

[0072] Preferably, the exonuclease is exonuclease III.

[0073] Various embodiments as described in the present detailed description can be combined according to the present invention unless clearly specified. [0074] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0075] LEGENDS OF THE FIGURES

[0076] Fig. 1 shows an exemplary workflow of the method of the invention. The phage genome is amplified by PCR into fragments of 12 kbp or less with overlapping DNA sequences. Gene addition (green), mutation (orange), and deletion (red dotted line) are introduced at any permissive locus. The PCR products are cleaned up and annealed in vitro. The DNA assembly reaction is directly added to a CFE reaction to produce phages. Engineered phages are propagated and titrated on the desired host and directly used for NGS sequencing and downstream applications.

[0077] Fig. 2 shows the cell-free synthesis of deGFP. a. Schematic showing the TXTL workflow used in this work. Reactions were incubated on well plates. deGFP (a slightly shorter version of eGFP1 ) was synthesized either through the P70a promoter or through the T7 transcriptional activation cascadel 2. b. Endpoint cell-free synthesis of deGFP as a function of plasmid concentration (P70a- degfp) and examples of kinetics, c. Endpoint cell-free synthesis of deGFP as a function of plasmid concentration (P70a-T7rnap fixed at a concentration of 0.2 nM, T7p14-degfp) and examples of kinetics.

[0078] Fig. 3 shows the cell-free synthesis of T7 WT from its genome at 0.1 nM. a. Schematic of the experiment. The T7 WT was titrated by the spotting assay. The TXTL reactions were diluted in Luria broth by factors of ten. 3.5 pl of the dilution were spotted on a lawn of E. coli B cells from left to right. The first spot on the left corresponds to a dilution of 10 of the TXTL reaction. The last spots show single plaques, which enable determining the PFU/ml. The whole experiment was repeated three times and spotted three times (nine rows for each replicate), b. replicate 1 . c. replicate 2. d. replicate 3.

[0079] Fig. 4 shows the T7 WT genome reconstruction and gene insertion by an embodiment of the method of the invention, a. Designated four DNA fragments for T7 WT Genome assembly: T7A (1 - 10700), T7B (10700-20500), T7C (20500-30600), T7D (30600-40000). The Table shows the primers and templates used for phage assembly, b. mCherry reporter gene insertion loci downstream of gene: 1 (T7 RNA polymerase), 10 (capsid protein), or gene 17 (tail fiber). The Table shows the primers and templates used for phage assembly c. T7-mChery life cycle d. Kinetics of infection of E. coli B cultures by T7-mCherry phages. Left: GD600 (dark curves). Right: fluorescence at 625 nm (red curves), arbitrary units.

[0080] Fig. 5 shows an exemplary implementation of the method of the invention to assemble the T7 WT phage. The T7 WT phage was assembled from four DNA fragments T7A (10.7 kbp), T7B (9.9 kbp), T7C (10.1 kbp), T7D (9.4 kbp) to final equimolar mix at 0.1 nM. The experiment was repeated three times and spotted three times for each repeat. Three different batches of fragments were used. DNA fragments in repeats a and b were amplified from T7 genomic DNA (used at a concentration <1 fM), in repeat c the four DNA fragments were amplified by adding phages from a clarified phage lysate to the PCR mixed. The first spot on the left corresponds to a dilution of 10. Negative controls consisted of assembling the T7 WT from only three of the fragments. The experiment was repeated three times. Each repeat used a different batch of DNA fragments. DNA fragments in repeats d and e were amplified from T7 genomic DNA, in repeat f the four DNA fragments were amplified by adding phages from clarified phage lysate to the PCR mixed. No phages were detected. Some slight inhibition of E. coli growth was observed in some spots, especially for BCD. g. TXTL of four T7 genome parts in separate reactions and spotted on a lawn of E. coli B cells. Parts T7B, T7C and T7D show some inhibition activity. From left to right: each spot corresponds to a dilution (with water) by a factor of two of the TXTL reaction. The blank TXTL reaction (no DNA) does not show any inhibition on the lawn. h. SDS PAGE of the products of each genome part. The protein marker (M) shows the ladder masses in kDa. The blank reactions (bk) are TXTL reactions with no DNA added to them. The T7 WT shows two major bands at a size that corresponds to the capsid proteins 36.5 kDa and 41 .8 kda (products of genes 10A and 10B). The T7A part shows two proteins. One at a size of about 40 kDa, which corresponds to the protein kinase and/or the DNA ligase (products of gene 0.7 and gene 1 .3). The second band corresponds to a band of about 30 kDa, hypothetically corresponding to a single stranded DNA binding protein (product of gene 2.5). The T7B part does not show any prominent bands. The T7C part shows two proteins, which corresponds to the capsid proteins 36.5 kDa and 41 .8 kda (products of genes 10A and 10B). The T7D part does not show any prominent bands. For T7B, T7C and T7D, proteins are synthesized without the T7 RNA polymerase being produced.

[0081] Fig. 6 shows an exemplary implementation of the method of the invention to insert a mcherry gene cassette into the T7 WT after gene 10A, before the T7 terminator of gene 10A. This experiment was carried out using oligonucleotides not designed to be specifically orthogonal. Left: assembly of the five DNA fragments including the mcherry cassette. The first spot on the left corresponds to a dilution of 10 of the TXTL reaction after the workflow. Right: assembly of the four DNA fragments without the mcherry cassette. Although resulting in a PFU/ml one thousand time smaller, a significant number of phages are assembled without the mcherry gene. After this result, we demonstrated that using orthogonal oligonucleotides for the DNA assembly eradicates leaky synthesis of phages, thus creating a leak-free workflow.

[0082] Fig. 7 shows a selection of T7-mCherry phages on plate reader. The method of the invention was used without orthogonal oligonucleotides to insert a mcherry gene cassette into the T7 WT after gene 10A, before the T7 terminator of gene 10A, as shown in Fig. S7. A serial dilution of the TXTL reaction expressing and synthesizing the T7-mCherry phages was added to E. coli B cultures to isolate phages carrying the mcherry gene cassette. With a factor of one thousand greater than T7 WT, T7-mCherry phages were isolated by taking the last dilution that shows mCherry fluorescence. The kinetics of the OD600 and fluorescence shows one T7-mCherry phage variant after selection.

[0083] Fig. 8 shows the implementation of the method of the invention with orthogonal oligonucleotides to insert a mcherry gene cassette into the T7 WT after gene 1 , gene 10A, and gene 17. a. Insertion after gene 1 . b. Insertion after gene 10A. c. Insertion after gene 17. The PFU/ml indicate the efficacy of the insertion, which depends up to a hundred-fold on the loci in this experiment. This experiment was replicated three times (three rows). The first spot on the left corresponds to a dilution of 10. d. The controls consisted of the same experiment without including the mcherry gene cassette in the DNA assembly. Negative controls were repeated three times. No phages were synthesized.

[0084] Fig. 9 shows an infection kinetic assay of T7-mCherry phages (assembled with orthogonal oligonucleotides) on E. coli B cultures. The left axis shows the OD600 (dark curves), the right axis shows the fluorescence at 625 nm (red curves), a. T7 WT. b. T7-mC-g1 -o. c. T7-mC-g10-o. d. T7- mC-g17-o.

[0085] Fig. 10 shows a T7 WT genome size reduction using the method of the invention, a. Assayed six deletions and combinations thereof. Primers and templates used for T7-mini assembly b. Schematic mapping of attempted deletions (black) on T7 WT and of T7-mini. c. Image of the plate showing the spots of a 10-fold serial dilution of 10⁸ PFU/mL T7 WT (left) and T7 mini (right) lysates on an E. coli B lawn after 2h of incubation at 37 °C.

[0086] Fig. 11 shows a schematic of the LPS and the ones recognized by T7 WT (boxed). Note that E. coli B is a RbLPS type.

[0087] Fig. 12 shows an in vitro genome ejection assay using purified LPS. Spotting (on a lawn of E. coli B) of serial 10-fold dilutions of the T7 WT and a T7-rfaD-1 phages. The two phages at -109 PFU/mL were pre-incubated with purified LPS at 0.2 mg/mL at 37 °C for 2 hours. RaLPS is the full size LPS. KLA is a ReLPS. In the case of T7 WT, only the RaLPS induces ejection of the genomes during the pre-incubation, resulting in no plaques. In the cases of T7-ReLPS both the RaLPS and the ReLPS (KLA) induce ejection of the genome during the pre-incubation resulting in no plaques. The first spot on the left corresponds to no dilution. The titer loss of T7-rfaD-s compared to T7 WT in presence of ReLPS shows that T7-rfaD-1 is -10 000 times more selective to ReLPS in vitro than T7 WT at 0.2 mg/mL ReLPS 37 °C, 2h. Each condition corresponds to tree individual phage/LPS mixture spotted once.

[0088] Fig. 13 shows an implementation of the method of the invention to engineer a T7-compilation genome, a. The T7-compilation genome incorporates three edits: deletions (del 2, 3, 4), insertion of the mcherry gene after gene 10, and mutation of the tail fiber to infect RePLS strains, b. Kinetics of infection of E. coli mutant strain rfaC cultures by the T7-compilation phage. Left: OD600 (dark curves). Right: fluorescence at 625 nm (red curves), c. Microscopy image of E. coli mutant strain rfaC infected with the T7-compilation phage (merge of phage contracts and fluorescence channels).

[0089] Fig. 14 shows an in vitro genome ejection assay using purified LPS. Spotting (on a lawn of E. coli B) of serial 10-fold dilutions of the T7-compilation phage generated by the method of the invention. The phage at 10⁸ PFU/mL was pre-incubated with purified LPS at 0.2 mg/mL. RaLPS is the full size LPS. KLA is a ReLPS. In the case of the T7-compilation, both the RaLPS and the ReLPS (KLA) induce ejection of the genome during the pre-incubation resulting in no plaques for RaLPS and a few undefined plaques on the first spot for ReLPS. The first spot on the left corresponds to no dilution.

[0090] Fig. 15 shows a spotting assay of three other phages after TXTL. Top: plaque assay of the E. co// phage VpaE1 on a lawn of E. coli B. Middle: spotting assay of the E. co// phage T6 on a lawn of E. coli B. Bottom: plaque assay of the Salmonella phage FelixOl on a lawn of Salmonella LT2. No plaques were observed when the genomic DNA of the three phages was spotted.

EXAMPLES

Example 1 : materials and methods

[0091] Reagents. The genomic DNA of phage T7 was purchased from Boca Scientific (# 310025). The DNA ladder for DNA electrophoresis was purchased from Invitrogen (10-787-018). The exonuclease III was purchased from NEB (#M0206S). DNA oligonucleotides were ordered from Integrated DNA Technologies (IDT), standard desalting. The bacterial strains were obtained from various sources as described in Table 2. Plasmid DNAs were obtained as follows: pTXTL-P70a- deGFP (Arbor Bioscience, #502056), pTXTL-T7p14-deGFP (Arbor Bioscience, #5021 1 1 ), pTXTL- P70a-T7rnap (Arbor Bioscience, #502082), pTXTL-T7p14-mmCherry (Arbor Bioscience, #502141 ). The LPS EH100 Ra (L9641 ) mutant and smooth LPS from E. coli 01 1 1 :B4 (L5293) were purchased from Sigma-Aldrich. The ReLPS was purchased from Avanti Polar Lipids (Kdo2-Lipid A (KLA), 699500). The phage lysates were sterilized with 0.22 pm centrifuge filter tubes (Costar #8160). The phage serial dilutions were performed with filter tips (Dutscher, #014210, #014220). The phage kinetics were performed in flat bottom transparent 96 bacterial culture well-plates with lids (Thermo Scientific Optical-Bottom Plates, #265301 ) on a Synergy H1 multi-mode microplate reader (Agilent). Phage spotting was performed on square Petri dishes (Greiner Bio-One #688102). Microscopy was done with an Olympus 1X81 inverted epi-fluorescence microscope mounted with a thermoplate (Tokai Hit). An imaging spacer (Grace Bio-Labs #654006) and microscopic slides (Fisher, #12-550-A3), and cover slides (Fisher #12542C) were used for bacterial lysis microscopic experiments. The purchased T7 genome (39.9 kbp, GenBank V01 146.1 ) was verified by NGS observing two mutations compared to GenBank V01 146.1 : (i) insertion of an A base after position 1896 in the gene 0.7; (ii) A to G mutation at position 22629 (N227S) in gene 9.

[0092] Cell-free transcription-translation. Cell-free gene expression was carried out using an E. coli TXTL system described previously (Sun et al, 2013, Garenne et al, 2021 ) with one modification. The strain BL21 -ArecBCD Rosetta2 was used, in which the recBCD gene set is knocked out to prevent the degradation of linear DNA (Batista et al). The preparation and usage of the TXTL system were the same as reported before (Sun et al, 2013, Garenne et al, 2021 ). Briefly, E. co// cells were grown in a 2xYT medium supplemented with phosphates. Cells were pelleted, washed, and lysed with a cell press. After centrifugation, the supernatant was recovered and preincubated at 37 °C for 80 min. After a second centrifugation step, the supernatant was dialyzed for 3 h at 4 °C. After a final spin-down, the supernatant was aliquoted and stored at -80 °C. The TXTL reactions comprised the cell lysate, the energy and amino acid mixtures, maltodextrin (30 mM) and ribose (30 mM), magnesium (2-5 mM) and potassium (50-100 mM), PEG8000 (3-4%), water and the DNA to be expressed. The reactions were incubated at 29 °C, in either 1 .5 ml tubes or on 96 well plates. For phage titration, the TXTL reactions were diluted with Luria broth (LB).

[0093] DNA amplification. The Q5 high-fidelity PCR polymerase (NEB #M0491 ) was used to amplify the fragment tail fiber fragment E0 (gene 771 146-1662, gp17 382-554). PCR mutagenesis was carried out with the Agilent Genemorph II Random Mutagenesis Kit (#200550) according to manufacturer instructions. Low (E1 ), medium (E2), and high (E3) mutation rates were obtained by adding respectively 500 ng, 50 ng, and 0.5 ng of E0 in the initial PCR mix (50 pl) and performing 30 PCR cycles. The PCR fragments for the assembly were otherwise amplified with KOD OneTM PCR Master Mix (No. KMM-101 ) according to manufacturer instructions. Either 1 pL of 1 ng/pL of T7 genome DNA or 1 pL of 10⁵ PFU/ml of clarified phage lysate obtained from a single plaque was used as template DNA. All PCR reactions were purified with a PCR clean-up kit (Invitrogen™ K310001 ) and DNA concentration was normalized to 20 nM in deionized water.

[0094] DNA assembly and TXTL reactions. Purified PCR fragments were mixed at an equimolar concentration of 0.5-5 nM. An equal volume of a 2x assembly mix (20 mM Tris-HCI pH 7.9, 100 mM NaCI, 20 mM MgCI2, 10% (w/v) PEG8000, 2 mM Dithiothreitol, 1 U/pL Exonuclease III) and 2.5 pL DNA mix were mixed on ice. Exonuclease inactivation was done by incubation of the DNA assembly reaction at 75 °C for 1 min. Single-stranded ends annealing was done by incubation at room temperature for 5 min. The mix was directly added to a TXTL reaction. Typically, 2.5 pL of the assembly mix was added to 7.5 pL of TXTL reaction. Negative controls were done by mixing all the PCR fragments except one replaced by an equal volume of water. For example, T7 genomic DNA was amplified in four fragments of 10.7 kbp ( T7A), 9.9 kbp ( T7B), 10.1 kbp ( T7C), and 9.4 kbp (T7D) using four sets of oligonucleotides with 50 bp overlapping sequences. The fragments were individually amplified by PCR and purified using standard procedures. For the PCR amplification, a few picograms of genomes or a few microliters of a clarified phage lysate are added directly as a template to the PCR mixture. The size of each fragment was verified by standard DNA gel electrophoresis and their concentration was measured by spectrophotometry. The fragments were mixed at an equimolar concentration in the nanomolar range, treated with the E. co// exonuclease III and annealed. The exonuclease, rapidly deactivated during the procedure, produces single-stranded gaps at the 3’ termini of the overlapping fragments enabling the annealing of complementary fragments. 2.5 pl of T7 DNA assembly reaction were added to a 7.5 pl TXTL reaction to express the annealed genomes. T7 phage genomes assembled from four parts were confirmed by NGS (T7- 4PCS).

[0095] NGS. Sequencing of the mutagenic PCR fragments was done by NGS (Illumina). DNA samples were converted to Illumina sequencing libraries using Illumina’s Truseq NanoDNA Sample Preparation Kit (Cat. # 20015964). During library creation, amplicon DNA was end-repaired with the adaptors, and indexes were ligated to each sample. The libraries did not undergo any PCR cycling. The final library size distribution was validated using capillary electrophoresis and quantified using fluorimetry (PicoGreen) and Kapa q-PCR. Pooled libraries were denatured and diluted to the appropriate clustering concentration. The libraries were then loaded onto the MiSeq paired-end flow cell and clustering occurred onboard the instrument. Once clustering was complete, the sequencing reaction immediately began using Illumina’s 4-color SBS chemistry. Upon completion of read 1 , 2 separate 8 or 10 base pair index reads were performed. Finally, the clustered library fragments were re-synthesized in the reverse direction thus producing the template for paired end read 2. Base call (.bcl) files for each cycle of sequencing were generated by Illumina Real Time Analysis (RTA) software. Primary analysis and de-multiplexing were performed using Illumina’s bcl2fastq software 2.20. The result of the bcl2fastq workflow was de-multiplexed in FASTQ files. Reference mapping of the obtained reads and variant calling were done with open-source Galaxy software (BWA-MEM and iVar, and quality Q > 30 were used). Nucleotide mutations were analyzed with a Python program.

[0096] Phage spotting assay. 1 .5% agar-LB plates were pre-incubated at 37 °C for 1 h. 10 mL of 0.7% soft agar was kept at 55 °C in a water bath. 100 pL of overnight bacterial culture were mixed with the soft agar and vortexed gently. The soft agar was slowly dispensed onto the agar LB plates plate to cover uniformly the entire surface of the agar plate. The soft-agar plates were left at room temperature for 15 min on a flat surface to solidify. Serial ten-fold dilutions in LB of either cell-free phage reaction or clarified phage lysates were prepared in 200 pL. Spotting: for each phage dilution, 3.5 pL were dropped onto the soft agar. For negative control TXTL reactions, the whole reaction was diluted in LB at a final volume of 25 pL and spotted in one droplet onto the soft agar layer. After spotting, the plate was left for 15 min on the bench to let droplets absorb onto the soft agar. The plates were incubated at 37 °C, facing down, for 4 h. Plaques were counted at the dilution where 1 <#plaques<20 per spot. Titers were typically calculated from three serial dilution spotting, plate uncertainties are estimated at the time of counting (duplication or plate belonging to the spot). For calculations on PFUs, Uncertainties were propagated using Python module Uncertainties: a Python package for calculations with uncertainties, Eric O. Lebigot.

[0097] Phage infection kinetic assay. Infection kinetics were carried out in 96 well plates in a Synergy Hi m microplate reader (Agilent). In each well, 180 pL of the host culture in LB (initial GD600 0.01 -0.04) were mixed with 20 pL of different serial phage dilutions. Each condition was replicated in four different wells. Positive controls consisted of 180 pL hosts in LB + 20 pL LB. Optical density at 600 nm and fluorescence intensity (excitation 580 nm, emission 610 nm for mCherry) was blanked against wells containing 200 pL of LB at each timestep. A lid was added to the 96 well plates to reduce evaporation during acquisition. The microplate reader was set to 37 °C with continuous double orbital shaking at 200 rpm. Optical density and fluorescence intensity were measured in each well every 3 min during 5-10 h. The mean and standard deviation of each condition were calculated at each timestep.

[0098] Microscopy. 100 pL of 2% agar were poured onto a slide with a spacer, covered with a cover slip, and let to solidify at 4 °C 1 h. 2 pL of host cells (OD600 ~ 0.2) and 2 pL phage lysate ~10⁵ PFU/ml were added on the agar pad. The agar slide was incubated at 37 °C for 15 min to allow cells to decant on the agar layer. A slide was added on top of the agar layer and cell growth and lysis were recorded on an epifluorescence microscope (40x objective). Time Lapse movies were assembled by recording one image every 2 min at 37 °C. Image composites and sequences were obtained with Imaged.

[0099] LPS-phage in vitro assay. LPS stock solutions were prepared in deionized water at 1 mg/ml and sonicated at 60 °C 30 min. T7 clarified lysates (10⁷-10¹⁰ PFU/ml) were mixed with LPS (final 200 pg/ml) in a final 50 pL volume and incubated at 37 °C 3 h. The phage LPS mixtures were serially diluted in LB and spotted on E. coli B lawn. For the p/G experiment, ReLPS was used at 400 pg/mL and incubated at 37 °C overnight to inhibit the potential remaining ReLPS+ phage phenotypes.

Example 2: phage synthesis workflow.

[0100] The T7 genome was re-assembled from long PCR fragments (<12 kbp) with overlapping sequences using a cheap assembly mix containing only an exonuclease, followed by heat inactivation of the enzyme. Annealed fragments are directly expressed in TXTL without additional steps enabling the synthesis and selection of T7 phage variants that integrate gene addition, deletion, and mutation. The workflow, achieved in under one day, delivers phages at titers comparable to titers obtained from bacterial lysate (10¹⁰'¹¹ PFU/ml) (Fig. 1 ).

Example 3: Cell-free transcription-translation.

[0101] The myTXTL system (Sun et al, 2013, Garenne et al, 2021 ) uses the endogenous E. coli core RNA polymerase and sigma factor 70 present in the lysate as the sole primary transcription proteins. This system does not contain any remaining live E. coli cells. Genes are expressed either from plasmids or linear dsDNA. In this work, all the TXTL reactions were carried out in batch mode at the scale of 1 -10 pl, either in 1 .5 mL tubes or in well plates. In batch mode, 80-100 pM of deGFP protein are produced after 12 h of incubation (Fig. 2) from an E. co// promoter (P70a (Garenne et al, 2019)). With the bacteriophage T7 promoter, 100-120 pM deGFP proteins are produced in 3-6 h. The wildtype phage T7 (T7 WT) is synthesized from its genome at a concentration of 10¹⁰-10¹¹ PFU/ml (plaque-forming units per milliliter) after 3 h of incubation (Fig. 3).

Example 4 :Plasmid construction and T7 WT rebooting.

[0102] A simple plasmid was first constructed to test the DNA assembly method. Two PCR fragments with complementary overhangs, the DNA encoding for T7 capsid gene 10, and the ColE1 plasmid backbone were assembled and transformed into E. coli cells. The plasmid was successfully recovered without any mutations. The T7 WT phage was then re-assembled from four PCR amplified fragments (Fig. 4a) with 50 base pairs (bp) overlaps. The four DNA parts were mixed at an equimolar concentration in the nanomolar range and annealed. The assembly reaction was directly added to the TXTL reaction to obtain T7 WT 10¹⁰ - 10¹¹ PFU/ml titers, similar to performing TXTL with a T7 WT genome concentration of 0.1 nM (Fig. 5a-c). Importantly, no phages were detected in control experiments expressing any combinations of only three annealed DNA fragments (Fig. 5d-f). Unexpectedly, some fragments expressed T7 components that inhibit E. coli growth in a dosedependent manner (Fig. 5g, h). This result demonstrates the ability of the workflow to explore the activity encoded by the products of genome gene sets.

Example 5: Leak-free genomic insertions.

[0103] Next, the method was used to insert the mCherry reporter gene cassette, consisting of a T7 promoter, a strong RBS, the mCherry fluorescent reporter gene, and a T7 terminator at three different T7 genomic loci (downstream of gene 1, downstream of gene 10 and of gene 17; Fig. 4b) to create three T7-mCherry phages. Five gene fragments were amplified using five sets of oligonucleotides designed with overlapping sequences matching the phage T7 and the mCherry cassette. TXTL of the in vitro assembled T7-mCherry genomes yielded a mixture of T7 WT phages and T7-mCherry phages in a ratio of ~1 :10³ phages (Fig. 6). mCherry-positive phage strains were recovered by screening in 96-well liquid culture format (Fig. 7), followed by fluorescence microscopy and NGS sequencing (T7-mC-g10).

The workflow was further improved to eliminate the synthesis of non-edited T7 phages and create a leak-free workflow by adopting a library of orthogonal oligonucleotides (Subramanian et al). Sense and antisense oligonucleotides were added to the 5’ and 3’ of the fragments to be annealed. With this design, all the phages detected were harboring the cassette, and no phages were synthesized in the control experiment without the insert (Fig. 8), mCherry fluorescence signal was detected upon lysis for the three T7-mCherry phages (Fig. 4c, d, Fig. 9) and insertion at the three loci was confirmed by NGS. This primer design approach was employed subsequently and systematically observed no leak in all the control experiments for any type of edits.

Example 6 : T7 phage genome size reduction.

[0104] The T7 WT capsid volume <2 kb limit on new gene insertion (Chan et al, Hausmann et al) can be relieved by deleting non-essential genome sequences. Here, the method described herein was used to reduce the T7 genome length while keeping phage infectibility on its natural host E. coli B. Six potential gene segments were targeted in the first 20 kbp of the T7 genome to delete non- essential class I genes (early genes) and class II genes (DNA metabolism and replication) (Hausmann et al) (Fig. 10a, Table 1). Class III structural genes, considered essential, were not targeted for deletion. The T7 genome was divided into the eight fragments (F1 to F8) adjacent to the six deletions (del-1 to del-6) (Fig. 10b) that were PCR-amplified with 20 bp-long orthogonal overhangs to bridge the fragments around the deleted regions. With this single design, any combination of deletions could be tested. A round of single deletion workflow led to viable phages for del2-4 (but not for del 1 , 5, and 6; Fig. 10a) that were combined in a second round to create a viable 35.8 kbp T7 (‘T7-mini”). In T7-mini, the T7 ligase (in de!2) is deleted, indicating that the ligase is not essential for DNA assembly in TXTL. The plaques formed by T7-mini are clear and circular (Fig. 10c). The three deletions were confirmed by whole genome sequencing. By releasing 4 kbp from the original WT genome of 39.9 kbp, the T7-mini could be re-engineered with insertions of up to 5 kbp.

[0105] Table 1 : List of the T7 phage genes and positions, with their functions when known and the size of the proteins (from GenBank V01 146.1 ).

Example 7: Expanding T7 phage host range.

[0106] Expanding the host range of phages is a major goal of phage engineering (Lemire et al), serving to unravel their infective mechanism and ecology, and to evolve potent phages for therapeutic and biotechnological applications. Here, the workflow was used to create and select variant T7 phages capable of infecting lipopolysaccharide (LPS)-variant ReLPS E. coli strains that are not infected by T7 WT.

[0107] Rough LPS are the primary and only receptor necessary for T7 WT infection of the natural host E. coli B. LPS consists of a lipid A, located in the outer lipid membrane, covalently linked to a polysaccharide chain. The hydrophilic polysaccharide chain consists of a core chain and a repeating O-specific chain. Rough LPS are classified by size from the largest RaLPS to the smallest ReLPS (Fig. 11 and Table 2). Different side chain modifications of the LPS can also be found in different E. coli strains (Raetz et al). Eleven E. coli genes are essential for T7 infection, from which nine are implicated in LPS biosynthesis, their deletions giving rise to truncated LPS forms (Qimron et al). T7 WT's ability to infect these mutants was verified, using a smooth LPS (Seattle 1946, smooth 06 (Frontiers, the Role of Outer Membrane Proteins and Lipopolysaccharides for the Sensitivity of Escherichia coli to Antimicrobial Peptides.) and ClearColi (Mamat et al) strains devoid of LPS as negative controls. T7 WT was observed to be unable to infect the negative controls, and three of the knockout strains, all harboring the smallest ReLPS (rfaC, IpcA, and rfaE, the efficiency of plating (EOP) <10^-5). As expected (Huss et al), rfaD strain had a higher EOP of 4 x 10^-4. Variable T7 WT EOP was recorded on E. coli B and the other knockout strains as reported in Table 2.

Table 2: Infection of T7 WT on E. coli strains

[0108] Previous work identified a T7 mutant phage capable of infecting ReLPS E. coliwhWe retaining its original LPS phenotype with mutations in the tail genes 11 and 12 and in the tail fiber gene 17 (Holtzman et al) ⁵⁰. Concordantly, a plaque was selected from a natural T7 WT phage variant infecting rfaD E. co// strain (T7-rfaD-1 ) harboring two mutations (G784E, in the tail gene 12; S541 R, in the tail fiber gene 17). T7-rfaD-1 was able to infect the other LPS knockout strains (rfaC, IpcA, and rfaE) as well as the natural host E. coli B strain. It was verified that the T7-rfaD-1 variant interacts with ReLPS with an in vitro assay. 10⁹ PFU/mL lysates of T7 WT or T7-rfaD-1 phages was incubated with either purified water, RaLPS, smooth LPS, or ReLPS lipopolysaccharide variants at 0.2 mg/ml (Fig. 12). The Phage-LPS mixtures were then titrated on E. coli B to reveal the remaining phages. As expected, T7 WT titers remained unchanged following pre-incubation with water, smooth LPS, and ReLPS. Concomitantly, no phage was detected following preincubation with RaLPS indicating that all the T7 WT phages ejected their genomes upon reaction with RaLPS (Gonzalez-Garcia et al). In contrast, no phage was detected following pre-incubation of T7-rfaD-1 with purified ReLPS or RaLPS (Fig. 12). The T7-rfaD-1 variant was estimated to be at least ~10 000 times more sensitive to ReLPS in vitro than T7 WT. This confirms that T7-rfaD-1 infects rfaD, rfaC, rfaE, and IpcA strains through interaction with ReLPS receptors. This assay provides credence for quantifying phage ReLPS infectious phenotypes in vitro.

Example 8: Compilation.

[0109] To demonstrate rapid, iterative use of the workflow, T7-compilation was assembled in one step-with the minimal genome (T7 mini), mCherry insertion downstream of gene 10, and tail fiber ReLPS infective mutation (Fig. 13a) using the corresponding PCR fragments. The resulting phages were confirmed by NGS sequencing and phenotyping (Fig. 13b-c, Fig. 14). Lysis kinetics of T7- compilation was slower as compared to the ReLPS infective phage indicative of an associated fitness cost. Example 9: Synthesis of other phages

[0110] Other phages than the T7 phage are targets of interest for the synthesis method described in the present disclosure. The cell free TXTL synthesis as described herein was successfully used to synthezise 3 other obligate lytic phages : T6, VPAE1 (Simoliunas et al), and FelixOl (Whichard et al) (Fig. 15).

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Claims

Claims [Claim 1] A cell-free method for synthesizing bacteriophages, comprising: (a) in vitro assembling overlapping DNA fragments into a phage genome, in a ligase-free assembling reaction, comprising:

(a1 ) digesting the overlapping DNA fragments by an exonuclease, and

(a2) annealing the overlapping DNA fragments to assemble a phage genome; and

(b) cell-free transcribing and translating the phage genome.

[Claim 2] The method of claim 1 , comprising a step of inactivating the exonuclease after step (a1 ) and before step (a2), preferably wherein the inactivation is by heat treatment.

[Claim 3] The method of claim 1 or claim 2, wherein step (a) is preceded by a step of amplifying a template phage genome by PCR to produce the overlapping DNA fragments.

[Claim 4] The method of any of the preceding claims, wherein the DNA fragments comprise overlapping DNA sequences of at least 5 bp at their 3’- and 5’-ends.

[Claim 5] The method of any of the preceding claims, wherein the overlapping DNA fragments comprise orthogonal overlapping sequences at their 3’- and 5’-ends such that two adjacent DNA fragments specifically anneal together.

[Claim 6] The method of any of the preceding claims, wherein the exonuclease is a 3’->5’ exonuclease, preferably is exonuclease III.

[Claim 7] The method of any of the preceding claims, wherein the exonuclease is the sole enzyme used in step (a).

[Claim 8] The method of any of the preceding claims, wherein the phage genome is not purified between step (a) and (b).

[Claim 9] The method of any of the preceding claims, wherein the overlapping DNA fragments comprise one or more genomic insertions, deletions, mutations and/or permutations with respect to the template phage genome.

[Claim 10] The method of any of the preceding claims, wherein the overlapping DNA fragments comprises one or more heterologous nucleotide sequences, in particular one or more heterologous genes.

[Claim 11] The method of any of the preceding claims, wherein step (b) comprises contacting the phage genome with an E coli lysate, preferably comprising the core RNA polymerase and sigma factor 70.

[Claim 12] The method of any of the preceding claims, wherein the phage is selected from T7, T6, T5, lambda, VPAE1 , and FelixOl .

[Claim 13] The method of any of the preceding claims, wherein more than 10⁹’ PFU/ml in particular more than 1 O¹⁰ PFU/ml phages are produced.

[Claim 14] The method of any of the preceding claims, wherein the synthesized genome comprises at least 10 kbp nucleotides, preferably at least 30kbp nucleotides.

[Claim 15] The method of any of the preceding claims, wherein the synthesized genome contains 2 assembly errors or less per junction, preferably no assembly error per junction.