WO2017055514A1 - Dna modification - Google Patents

Abstract

The invention relates to the field of genetic engineering, and to the modification of nucleic acids and organismal genomes in particular. Specifically, the invention concerns the chemical modification of nucleic acids for the improved transformation of cells. Accordingly, the invention provides for modified nucleic acids, methods of modifying nucleic acids, methods of transforming cells and methods for the sequence-directed site-specific genetic modification of cells. The invention additionally encompasses cells transformed with modified nucleic acids and expression constructs for delivery and expression of modified nucleic acids. The nucleic acids, expression constructs, cells and methods of the invention find application in many areas of biotechnology, including, for example, genome editing.

Description

DNA MODIFICATION

FIELD OF THE INVENTION The present invention relates to the field of genetic engineering, and to the modification of nucleic acids and organismal genomes in particular. The present invention involves the chemical modification of nucleic acids for the improved transformation of cells. The invention concerns modified nucleic acids, methods used to achieve the chemical modification of nucleic acids and associated cells, expression constructs, bacteriophages and compositions. Additionally, the invention encompasses cells transformed with modified nucleic acids, expression constructs for delivery and expression of modified nucleic acids, nucleases and guide RNAs within cells, bacteriophages, compositions and methods for the sequence-directed site-specific genetic modification of cells.

BACKGROUND TO THE INVENTION

In the past decade, the discovery that the prokaryotic CRISPR adaptive immune machinery and in particular CRISPR-Cas can be repurposed to rapidly and precisely edit nucleic acid sequences at specific sites in a genome of interest has provided a useful set of tools for genetic engineering which has quickly become the gold standard method for genome editing.

One of the principle reasons for the widespread adoption of the technology is that the sequence-specific cleavage of genomic DNA by the CRISPR-Cas system is much simpler and more efficient when compared with alternative tools for sequence- specific genome manipulation, for example Zinc Finger Nucleases (ZFNs) or Transcription activator-like effector nucleases (TALENs) due to its two-component structure. The sequence-specific cleavage of genomic DNA by the CRISPR-Cas system is mediated by two distinct components; a guide RNA (gRNA) and an endonuclease, typically the CRISPR associated (Cas) nuclease, Cas9. The gRNA is a chimera of the endogenous bacterial CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), combining the targeting specificity of the crRNA with the scaffolding properties of the tracrRNA. In order to direct cleavage at different target sequences, it is only necessary to modify the targeting sequence of the gRNA, rather than requiring technically complex protein manipulation. The ease with which sequences of interest can be targeted with precision has meant that the technology has been rapidly adopted and the genomes of a range of cells and organisms have been modified using the CRISPR-Cas9 system.

However, one practical limitation which has constrained application of the technology is the degradation of the DNA repair template. In order to precisely generate specific sequence mutations or insertions in a gene of interest, a double strand break (DSB) is generated at the intended site using Cas9 directed by the gRNA. DNA repair templates are provided containing the desired sequence. The sequence-specific repair mechanism takes advantage of the endogenous Homology Directed Repair (HDR) mechanisms in target cells, for which a high degree of sequence similarity to the sequence immediately upstream and downstream of the intended editing site is required to ensure faithful and efficient repair of the DSB. A known limitation of this technique for DNA editing is that in situations where a Protospacer adjacent motif (PAM) sequence is located close to the intended editing site, it may be desirable (in the context of retaining as much sequence homology as possible to drive efficient HDR of the Double Strand Break (DSB) to include the PAM sequence in the repair template. However, due to the high degree of homology required between target and template, distinguishing one from the other is a challenge and the presence of the PAM site may result in Cas9-mediated cleavage (and subsequent degradation) of the repair template.

One view is that this may account for the inability to genetically transform some cells with the CRISPR Cas9 system. Currently, various methods of repair template design are employed to avoid this problem, such as ensuring that either the target sequence is not immediately followed by the PAM sequence or that the PAM sequence is either excluded or mutated to prevent further Cas9 targeting after the initial DSB is repaired.

However, these are template design rules aimed at avoiding repair template degradation rather than reliable technical solutions to the problem and which involve making compensatory alterations to the repair template which are unrelated to the desired base changes. Furthermore, target-template distinction remains problematic in situations where the goal is to introduce a very specific mutation or sequence alteration.

Modified bases are found in eukaryotes, prokaryotes, and bacteriophages (Gommers-Ampt and Borst, 1995, FASEB J. 9: 1034-1042) and have various biological functions. Modified bases in eukaryotes are thought to be principally involved in gene regulation (Munzel et al., 201 1 , Angew. Chem. Int. Ed. 50: 6460- 6468). For example in Trypanosoma brucei, a kinetoplastid flagellate, the modified DNA base b-D-Glucosyl-hydroxymethyluracil is thought to be involved in transcriptional repression of variant surface glycoprotein (VSG) genes (van Leeuwen et al., 1998, Mol Cell Biol 18: 5643-5651 ). Modified bases in prokaryotes on the other hand, mainly play a role in host-defence mechanisms, enabling differentiation between foreign and non-foreign DNA (Wilson and Murray, 1991 , Annu. Rev. Genet. 25: 585-627). In contrast, some bacteriophages have been shown to use modified bases to evade prokaryotic/bacterial restriction-modification (R-M) systems (Labrie et al., 2010, Nat. Rev. Microbiol. 8: 317-327). The model organism bacteriophage (phage) T4 has acquired a pathway to bypass the R-M systems of its host by incorporation and subsequent glucosylation of hydroxymethylcytosine (hmC) in its DNA (Flaks and Cohen, 1958, Federation Proc. 17: 220; Loenen and Raleigh, 2014, Nucleic Acids Res. 42: 56-69). Glucosylation protects the hmC DNA from cleavage by the modification dependent system (MDS) McrBC. In addition to a classical R-M system, E. coli also harbours a modification-dependent system (MDS), the McrBC enzyme, that is specific for hmC (Raleigh and Wilson, 1986, Proc. Natl. Acad. Sci. USA, 83: 9070-9074). Phage T4 is impervious to this MDS system because its hmC residues are glucosylated by transfer of glucose from uridine diphosphate glucose (UDPG) to hmC. Another way of overcoming MDS restriction is by production of ocr (overcome classical restriction) proteins, for instance as observed in phage T3 and T7 (Kruger et al., 1977, Mol. Gen. Genet. 153: 99-106), which competitively inhibit the binding of type I DNA restriction enzymes to their DNA target. Since the discovery that modified bases produced by bacteriophages function in the evasion of prokaryotic/bacterial restriction-modification (R-M) systems, a complicated and contradictory picture of the molecular functions of modified nucleotide residues has emerged and to date, any additional functions of these residues have remained unclear.

While others have previously reported that wild-type T4 containing glucosyl hydroxymethocytosine was insensitive to attack by CRISPR-Cas9 designed with spacer sequences targeting T4 in vivo, but T4 mutant phages with unmodified cytosine were sensitive (see Bryson et ai, 2015, mBio 6(3):e00648-15. doi:10.1 128/mBio.00648-15), in direct contrast it has been found that the chemical modification of cytosine residues does not protect DNA molecules from degradation by host cell nucleases in vivo (Yaung et ai, 2014, PLoS ONE 9(6):e9881 1 . doi:10.1371/journal.pone.009881 1 ).

The present invention is based on the discovery of the minimal genetic pathway required to convert cytosine residues to glucosyl hydroxymethylcytosine residues. Surprisingly, it was found that the introduction of polynucleotide sequences encoding enzymes which mediate the conversion of cytosine residues to glucosyl hydroxymethylcytosine residues into bacterial cells produces modified DNA which is impervious to degradation by host cell nucleases. In particular, it was found that the modification of cytosines in this way allowed the modified DNA to successfully evade the CRISPR-Cas9 and CRISPR-Cas3 surveillance systems. It has not previously been appreciated that genes encoding the enzymes responsible for producing glucosyl hydroxymethylcytosine might be expressed and functional in cells, nor has producing glucosyl hydroxymethylcytosine been proposed as a mechanism to reduce the degradation of repair template DNA and thus improve the reliability of DNA editing.

The present invention therefore provides the first prokaryote that substitutes cytosine (C) in its genome with hmC and glucosyl hydroxymethylcytosine (ghmC) and solves the technical problem of providing polynucleotides to cells which are resistant to or capable of avoiding the native cell nuclease enzymes. A practical application of the discovery of the invention is a novel tool for the production of modified DNA, that when introduced to a cell evades sequence-specific degradation by the cell, which improves the efficiency of genetic engineering of cellular DNA.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides a cell which has deoxycytidylate 5- hydroxymethyltransferase (dCMP Hmase) activity and deoxy-Nucleotide Monophosphate (dNMP) kinase activity, and wherein at least a proportion of the cytosine residues of the DNA of the cell are hydroxymethylcytosine (hmC) residues.

The deoxycytidylate 5-hydroxymethyltransferase (dCMP Hmase) enzyme (gene 42) preferably comprises an amino acid sequence of SEQ ID NO: 1 or a sequence of at least 75% identity thereto; or is encoded by a polynucleotide sequence of SEQ ID NO: 2 or a sequence of at least 75% identity thereto.

The deoxy-Nucleotide Monophosphate (dNMP) kinase enzyme (gene 1 ) preferably comprises an amino acid sequence of SEQ ID NO: 3 or a sequence of at least 75% identity thereto; or is encoded by a polynucleotide sequence of SEQ ID NO: 4 or a sequence of at least 75% identity thereto.

Advantageously, the present invention is of broad applicability and cells of the present invention may be derived from any genetically tractable organism which can be cultured. Cells of the invention may be prokaryotic or eukaryotic, providing that they are viable and capable of replication. For example they may be bacterial cells, archaeal cells, protist cells, fungal cells, plant cells, algal cells and animal cells including human cells (but not including embryonic stem cells).

Preferred cells for use in accordance with the present invention are commonly selected from species which exhibit high growth rates, are easily cultured and/or transformed, display short generation times and/or species which have established genetic resources associated with them. Preferably, cells of the invention are prokaryotic cells, more preferably bacterial cells; for example Escherichia coli cells.

Prokaryotic cells of the invention are preferably those free of phage infection, whether lytic or lysogenic. In other preferred embodiments, the prokaryotic cells of the invention are free of T-even phage infection. Exemplary embodiments of the invention include bacterial cells, for example E. coli cells which are free of T-even phage infection. In other exemplary embodiments, the cells are bacterial cells, for example E. coli cells, which are free of T4 phage infection.

Typically, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% at least 80%, at least 90% or at least 95% of the cytosine residues of the DNA of the cell are hydroxymethylcytosine (hmC) residues. Preferably, the proportion of the hydroxymethylcytosine residues of the DNA of the cell is at least 90%; preferably at least 98%. In some instances the proportion of residues is 100%.

As well as deoxycytidylate 5-hydroxymethyltransferase (dCMP Hmase) activity and deoxy-Nucleotide Monophosphate (dNMP) kinase activity, cells of the invention may also have glycosyl transferase activity. Glycosyltransferases are enzymes that catalyze the formation of the glycosidic linkage to form a glycoside. In the context of the invention, it will be understood that any glycosyltransferase enzyme may be used for this purpose. Preferably, those glycosyltransferase enzymes which utilise 'activated' nucleotide sugar phosphates as glycosyl donors, and catalyze the transfer of saccharide moieties from activated nucleotide sugar phosphates (also known as the "glycosyl donor") to a nudeophilic glycosyl acceptor molecule, the nucleophile of which can be oxygen- carbon-, nitrogen-, or sulphur-based, are employed.

Accordingly, at least a proportion of the cytosine residues of the DNA of cells having glycosyl transferase activity are glycosyl hydroxymethylcytosine residues. Typically, in cells having glycosyl transferase activity, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% at least 80%, at least 90% or at least 95% of the cytosine residues of the DNA of the cell are glycosyl hydroxymethylcytosine residues. In accordance with the invention any glycosyl transferase may be used. Preferably, however, cells of the invention have glucosyl transferase activity. It will be appreciated however, that depending on the desired characteristics of the DNA to be produced in the cell, an alternative glycosyl transferase might be used in place of or in addition to a glucosyl transferase, including, but not limited to for example, galactose-, mannose-, acetyl glucosamine-, fucose-, xylose-, rhamnose- or fructose- transferases. In cells having glucosyl transferase activity, at least a proportion of the cytosine residues of the DNA of the cell are glucosyl hydroxymethylcytosine (ghmC) residues. More preferably, in cells having glycosyl transferase activity, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% at least 80%, at least 90% or at least 95% of the cytosine residues of the DNA of the cell are glucosyl hydroxymethylcytosine residues.

Optionally, cells of the invention may have a glucosyl transferase activity and/or β glucosyl transferase activity. Where cells have both a and β glucosyl transferase activity, the relative contributions of a or β may be varied according to the desired characteristics of the DNA.

Cells of the invention may additionally have deoxycytidine-triphosphatase (dCTPase) activity. It will be understood that the enzyme conferring dCTPase activity on the cell may have other, additional functions or activities within the cell. Preferably, the enzyme will have dCTPase and deoxycytidine diphosphatase/deoxyuridine- triphosphatase (dCDPase/dUTPase) activity. The enzyme will preferably have dCTPase, dCDPase/dUTPase and dUDPase activity. More preferably, the dCTPase enzyme (gene 56) comprises an amino acid sequence of SEQ ID NO: 5 or a sequence of at least 75% identity thereto. Preferably, the dCTPase enzyme is encoded by a polynucleotide sequence of SEQ ID NO: 6 or a sequence of at least 75% identity thereto.

Usefully, the present invention also provides a genetically engineered cell, comprising a deoxycytidylate 5-hydroxymethyltransferase (dCMP Hmase) gene and a deoxy-Nucleotide Monophosphate (dNMP) kinase gene. Preferably, the deoxycytidylate 5-hydroxymethyltransferase (dCMP Hmase) gene comprises polynucleotide sequence of SEQ ID NO: 2 or a sequence of at least 75% identity thereto. Preferably, the deoxy-Nucleotide Monophosphate (dNMP) kinase gene (gene 1 ) comprises polynucleotide sequence of SEQ ID NO: 4 or a sequence of at least 75% identity thereto.

The genetically engineered cell may optionally further comprise a deoxycytidine- triphosphatase (dCTPase) gene. Preferably, the genetically engineered cell further comprises a glycosyl transferase gene. More preferably, the glycosyl transferase gene is a glucosyl transferase gene. The glucosyl transferase gene may be an a or β glucosyl transferase gene. Preferably, the glucosyl transferase is a beta glucosyl transferase (β-GT) enzyme comprising an amino acid sequence of SEQ ID NO: 7 or a sequence of at least 75% identity thereto (β-GT). Preferably, the glucosyl transferase encoded by a polynucleotide sequence of SEQ ID NO: 8 or a sequence of at least 75% identity thereto (β-GT).

Preferably, the deoxycytidine-triphosphatase (dCTPase) enzyme comprises an amino acid sequence of SEQ ID NO: 5 or a sequence of at least 75% identity thereto (GENE 56). Preferably, the deoxycytidine-triphosphatase (dCTPase) enzyme is encoded by a polynucleotide sequence of SEQ ID NO: 6 or a sequence of at least 75% identity thereto (GENE 56).

The percentage amino acid sequence identity with any of the aforementioned reference sequences (SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7) is determinable as a function of the number of identical positions shared by the sequences in a selected comparison window, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

A protein or polypeptide fragment of the invention may be characterised in terms of both the reference sequence (SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 9) and any aforementioned percentage variant thereof as defined by percentage sequence identity.

In all of the aforementioned aspects of the invention, where there is a reference polynucleotide or amino acid sequence and sequences of at least a certain percentage identity are disclosed, e.g. 75%, then optionally the percentage identity may be different. For example: a percentage identity which is "at least" one of the following: 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100%. Such sequence identity with a dCMP Hmase, dNMP kinase, dCTPase, or an or a or β glucosyl transferase amino acid sequence, gene or cDNA from, e.g. T4 phage, is a function of the number of identical positions shared by the sequences in a selected comparison window, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

In the polynucleotide sequences in accordance with the invention defined above, the nucleotide sequence may be that which encodes the respective SEQ ID NOs: 1 , 2, 3, 4, 5, 6, 7, 8 or 9 or in defining the range of variant sequences thereto, it may be defined instead as a sequence hybridizable to the reference nucleotide sequence, preferably under stringent conditions, more preferably very high stringency conditions. The term "stringent conditions" may be understood to describe a set of conditions for hybridization and washing and a variety of stringent hybridization conditions will be familiar to the skilled reader. Hybridization of a nucleic acid molecule occurs when two complementary nucleic acid molecules undergo an amount of hydrogen bonding to each other known as Watson-Crick base pairing. The stringency of hybridization can vary according to the environmental (i.e. chemical/physical/biological) conditions surrounding the nucleic acids, temperature, the nature of the hybridization method, and the composition and length of the nucleic acid molecules used. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed in Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001 ); and Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology— Hybridization with Nucleic Acid Probes Part I, Chapter 2 (Elsevier, New York, 1993). The Tm is the temperature at which 50% of a given strand of a nucleic acid molecule is hybridized to its complementary strand. In any of the references herein to hybridization conditions, the following are exemplary and not limiting:

Very High Stringency (allows sequences that share at least 90% identity to hybridize) Hybridization: 5x SSC at 65°C for 16 hours

Wash twice: 2x SSC at room temperature (RT) for 15 minutes each

Wash twice: 0.5x SSC at 65°C for 20 minutes each

High Stringency (allows sequences that share at least 80% identity to hybridize)

Hybridization: 5x-6x SSC at 65°C-70°C for 16-20 hours

Wash twice: 2x SSC at RT for 5-20 minutes each

Wash twice: 1 x SSC at 55°C-70°C for 30 minutes each

Low Stringency (allows sequences that share at least 50% identity to hybridize)

Hybridization: 6x SSC at RT to 55°C for 16-20 hours

Wash at least twice: 2x-3x SSC at RT to 55°C for 20-30 minutes each.

In all aforementioned aspects of the present invention, amino acid residues may be substituted conservatively or non-conservatively. Conservative amino acid substitutions refer to those where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not alter the functional properties of the resulting polypeptide. Similarly it will be appreciated by the skilled reader that nucleic acid sequences may be substituted conservatively or non-conservatively without affecting the function of the polypeptide. Conservatively modified nucleic acids are those substituted for nucleic acids which encode identical or functionally identical variants of the amino acid sequences. It will be appreciated by the skilled reader that each codon in a nucleic acid (except AUG; typically the only codon for methionine) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a polynucleotide or polypeptide, which encodes a polypeptide of the present invention, is implicit in each described polypeptide sequence.

The following table of exemplary amino acid substitutions is provided for guidance:

Table 1 : Exemplary conservative amino acid substitutions

Original Residue Exemplary Original Residue Exemplary

Conservative Conservative

Substitution Substitution

ALA SER LEU ILE; VAL

ARG LYS; GLN LYS ARG; GLN

ASN GLN; HIS MET LEU; ILE

ASP GLU PHE MET; LEU; TYR

CYS SER SER THR

GLN ASN; LYS THR SER

GLU ASP TRP TYR

GLY PRO TYR TRP; PHE

HIS ASN; GLN VAL ILE; LEU

ILE LEU; VAL

Preferably, a genetically engineered cell of the invention is a prokaryotic cell, usually a bacterial cell and commonly an E. coli cell. Preferably the cell is viable and capable of replication. More preferably, the cell is free of phage infection, particularly T-even phage infection.

Usually, the polynucleotide sequences encoding deoxycytidylate 5- hydroxymethyltransferase (dCMP Hmase), deoxy-Nucleotide Monophosphate (dNMP) kinase, deoxycytidine-tnphosphatase (dCTPase) and/or glycosyl transferase are comprised in a vector. Preferably the polynucleotide sequences encoding deoxycytidylate 5-hydroxymethyltransferase (dCMP Hmase), deoxy-Nucleotide Monophosphate (dNMP) kinase, deoxycytidine-triphosphatase (dCTPase) and/or glycosyl transferase are comprised in a plasmid. Advantageously, in situations where the genetically engineered cell further comprises a glycosyl transferase gene substantially all of the cytosine residues of the DNA of the cell are glycosyl hydroxymethylcytosine residues. In cells having glycosyl transferase activity, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% at least 80%, at least 90% or at least 95% of the cytosine residues of the DNA of the cell are glycosyl hydroxymethylcytosine residues. Where the glycosyl transferase gene is a glucosyl transferase, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% at least 80%, at least 90% or at least 95% of the cytosine residues of the DNA of the cell are glucosyl hydroxymethylcytosine residues. Preferably, substantially all of the cytosine residues of the DNA of the cell are glucosyl hydroxymethylcytosine (ghmC) residues. The expression of all or any of the aforementioned genes in a genetically engineered cell of the invention may be controlled by placing it under the control of a heterologous regulatory sequence, which is capable of directing the desired expression of the gene. Such regulatory sequences are nucleotide sequences which are capable of influencing the transcription or translation of a gene or gene product, for example in terms of initiation, accuracy, rate, stability, downstream processing and mobility. Examples of regulatory sequences envisaged for use in accordance with the present invention include promoters, 5' and 3' UTR's, enhancers, transcription factor or protein binding sequences, start sites and termination sequences, ribosome binding sites, recombination sites, polyadenylation sequences, sense or antisense sequences. They may be DNA, RNA or protein. The regulatory sequences may be bacteria-, plant-, fungus- or virus derived and optimally may be derived from the same species as the cell being engineered. Such regulatory sequences may suitably be placed 5' and/or 3' of the endogenous gene and may include, but are not limited to promoter sequences, terminator fragments, polyadenylation sequences or enhancer sequences operably linked to the polynucleotide sequences.

Preferably, in genetically engineered cells of the invention, comprising a deoxycytidylate 5-hydroxymethyltransferase (dCMP Hmase) gene and a deoxy- Nucleotide Monophosphate (dNMP) kinase gene, and optionally, additionally comprising a deoxycytidine-triphosphatase (dCTPase) gene and/or a glycosyl transferase gene, expression of the genes is under operative control of at least one promoter. Any suitable promoter which provides desirable expression levels in cells to be engineered may be selected. Accordingly, suitable promoters controlling the expression of these genes may be constitutive, whereby they drive expression under most environmental conditions and developmental stages (for example the T7 promoter, which originates from bacteriophage T7) or they may be inducible; initiating transcription in response to environmental, chemical or developmental cues, such as temperature, light, chemicals and/or other stimuli. Typically, selected promoters will have a high relative frequency of transcription initiation.

Suitable promoters may be heterologous, native or synthetic promoters. Preferably, theat least one promoter is an inducible promoter, for example a Rhamnose inducible promoter or a trc promoter. Preferably, expression of the genes is under operative control of a single promoter, which may for example be an inducible promoter for instance a Rhamnose inducible promoter or a trc promoter. More preferably, expression of the genes is under operative control of a Rhamnose inducible promoter.

Where an inducible promoter is chosen, transcription of the promoter will preferably be tightly-regulated, such that it has a minimal level of basal transcription. Where this is not the case, for instance in the case of the trc promoter, the basal level of transcription may be reduced by addition of additional regulatory elements downstream of the promoter region, for example a lac operator.

According to the present invention, for use in generating hmC or ghmC DNA, polynucleotide sequences coding for any of the aforementioned enzymes may be provided in an expression cassette comprising one or more regulatory sequences to modulate the expression of the relevant genes in a genetically engineered cell of the invention. Preferably, the regulatory sequences are located such as to be operably linked to the gene, in order that expression of the genes is directed in a desired manner in the cell. The polynucleotides as described herein, and/or a regulatory sequence are preferably provided as part of an expression cassette for expression of the polynucleotide in a cell. Suitable expression cassettes for use in the present invention are well known and may be constructed by standard techniques known in the art, to comprise 5' and 3' regulatory sequences, including, but not limited to promoter sequences, terminator fragments, polyadenylation sequences or enhancer sequences operably linked to the polynucleotide sequences. Such elements may be included in the expression construct to obtain the optimal functional expression of deoxycytidylate 5-hydroxymethyltransferase (dCMP Hmase), deoxy-Nucleotide Monophosphate (dNMP) kinase, deoxycytidine-triphosphatase (dCTPase) and/or glycosyl transferase in the cell.

In addition, polynucleotide sequences encoding, for example, reporters or selectable markers may be included as desired. Expression cassettes of the invention may also contain one or more restriction sites, in order to enable insertion of the polynucleotide sequence encoding the deoxycytidylate 5-hydroxymethyltransferase (dCMP Hmase), deoxy-Nucleotide Monophosphate (dNMP) kinase, deoxycytidine- triphosphatase (dCTPase) and/or glycosyl transferase and/or one or more regulatory sequences into the genome of the cell, at pre-selected loci. Transcription and translation initiation regions, to enable expression of the incoming genes, transcription and translational termination regions, and regulatory sequences may also be provided in the expression cassette. These sequences may be native to the cell being transformed, or alternatively may be heterologous. Expression cassettes of the invention may be capable of functioning (i.e. expression) in multiple, different cells.

Accordingly, the present invention provides a DNA expression construct comprising a promoter and a deoxycytidylate 5-hydroxymethyltransferase (dCMP Hmase) gene comprising a polynucleotide sequence of SEQ ID NO: 2 or a sequence of at least 75% percentage identity thereto and a deoxy-Nucleotide Monophosphate (dNMP) kinase gene comprising a polynucleotide sequence of SEQ ID NO: 4 or a sequence of at least 75% percentage identity thereto, wherein the genes are under operative control of the promoter. An expression construct may optionally further comprise a deoxycytidine-triphosphatase (dCTPase) gene comprising a polynucleotide sequence of SEQ ID NO: 6 or a sequence of at least 75% percentage identity thereto under operative control of the promoter. Preferably, an expression construct will further comprise a glycosyl transferase gene under operative control of the promoter. Preferably, the glycosyl transferase gene is a glucosyl transferase gene comprising a polynucleotide sequence of SEQ ID NO: 8 or a sequence of at least 75% percentage identity thereto.

In another aspect the present invention provides a DNA expression construct comprising a promoter and a gene desired to be expressed in a prokaryotic cell under operative control of the promoter, wherein at least a proportion of the cytosine residues of the DNA are hydroxymethylcytosine (hmC) or glycosyl hydroxymethylcytosine (ghmC); wherein the promoter is preferably a prokaryotic promoter. Accordingly, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% at least 80%, at least 90% or at least 95% of the cytosine residues of the DNA of the cell are hydroxymethylcytosine (hmC) or glycosyl hydroxymethylcytosine residues. Preferably, the proportion of cytosine residues that are that are hydroxymethylcytosine (hmC) or glycosyl hydroxymethylcytosine is at least 95%; preferably at least 98%; more preferably 100%. Preferably, at least a proportion of the cytosine residues of the DNA are glucosyl hydroxymethylcytosine (ghmC) residues. Accordingly, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% at least 80%, at least 90% or at least 95% of the cytosine residues of the DNA of the cell are glucosyl hydroxymethylcytosine (ghmC) residues. Genetically engineered prokaryotic cells of the invention may have a hydroxymethyl- selective restriction enzyme system active against hmC. In such circumstances the hydroxymethyl-selective restriction enzyme system active against hmC is preferably inactivated. More preferably the cell is an E. coli cell, which has a hydroxymethyl-restriction enzyme system active against hmC and the system is inactivated.

Even more preferably, the cell is an E. coli strain which is a mutant in which the methylation-requiring restriction system McrBC is inactivated. Such cells may include for example, but are not limited to E cloni® 10G, E. coli strain ER1821 , E. coli strain DH10B, E. coli strain T7 Express, ER1793, K803, C2523, or ER2925.

The present invention also provides bacteriophages, other than T-even phages, wherein at least a proportion of the cytosine residues of the phage DNA are hydroxymethylcytosine (hmC) or glucosyl hydroxymethylcytosine (ghmC). Preferably, the proportion of cytosine residues of the DNA of the bacteriophage that are hydroxymethylcytosine (hmC) or glucosyl hydroxymethylcytosine (ghmC) is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% at least 80%, at least 90% or at least 95% of the cytosine residues. More preferably, the proportion of cytosine residues that are hydroxymethylcytosine (hmC) or glucosyl hydroxymethylcytosine (ghmC) is at least 95%; even more preferably at least 98%; and still more preferably 100%. A bacteriophage of the invention may be provided as comprised in a composition. Preferably the present invention provides a composition comprising a bacteriophage, which is not T4 page, which comprises polynucleotides of SEQ ID NO: 2 and 4 or polynucleotide sequences of at least 75% identity thereto or functional fragments thereof. Optionally, the bacteriophage may further comprise a polynucleotide of SEQ ID NO: 6 or a polynucleotide sequence of at least 75% identity thereto or functional a fragment thereof. Optionally, the bacteriophage may further comprise a polynucleotide of SEQ ID NO: 8 or a polynucleotide sequence of at least 75% identity thereto or functional a fragment thereof. Preferably, the bacteriophage will comprise polynucleotides of SEQ ID NO: 2, 4, 6 and 8 or polynucleotide sequences of at least 75% identity thereto or functional fragments thereof. Even more preferably, an operon comprising polynucleotides of SEQ ID NO: 2, 4, 6 and 8 or polynucleotide sequences of at least 75% identity thereto or functional fragments thereof is genonnically inserted into a phage genome, which is not T4 phage, for example phage Lambda. Preferably, the operon will comprise the polynucleotide sequence of SEQ ID NO: 9 or a sequence of at least 75% identity thereto.

The present invention also provides methods of preparing DNA, wherein at least a proportion of the cytosine residues are hydroxymethylcytosine (hmC), comprising culturing a cell of the invention which comprises deoxycytidylate 5- hydroxymethyltransferase (dCMP Hmase) activity and deoxy-Nucleotide Monophosphate (dNMP) kinase activity for a period, isolating the cell and then extracting DNA from the cell. Furthermore the present invention provides a method of preparing DNA, wherein at least a proportion of the cytosine residues of the DNA are glycosyl hydroxymethylcytosine residues, comprising culturing a cell of the invention for a period, isolating the cell and then extracting DNA from the cell. Methods of isolating cells and of DNA extraction are well known in the art.

Preferably, at least a proportion of the cytosine residues of the DNA are glucosyl hydroxymethylcytosine (ghmC) residues.

Cells employed in the methods of the invention may be prokaryotic or eukaryotic, but are preferably prokaryotic cells. Cells employed in the methods of the invention are more preferably E. coli cells.

Depending on the type of cell used in accordance with the method and the desired application it is envisaged that DNA extracted from the cells may be genomic DNA, mitochondrial DNA, plastid DNA, chloroplast DNA or plasmid DNA. Preferably, DNA extracted from the cells is plasmid DNA.

The present invention also provides a method of preparing DNA having at least a proportion of cytosine residues as glycosyl hydroxymethylcytosine residues, comprising contacting isolated DNA having at least a proportion of cytosine residues as hydroxymethylcytosine (hmC) with a glycosyl transferase in the presence of a sugar. It will be appreciated that a number of sugars may be used. Usually, the sugar is one selected from e.g. an activated nucleotide sugar (ADP-glucose, UDP- glucose, UDP-a-D-GIc, UDP-a-D-Gal, UDP-a-D-GalNAc, UDP-a-D-GlcNAc, UDP-a- D-GIcA, UDP-a-D-Xyl, GDP-a-D-Man, GDP- -L-Fuc, CMP- -D-Neu5Ac, CDP- glucose and TDP-glucose), galactose, mannose, acetyl glucosamine, fucose, xylose, rhamnose or fructose. Preferably the sugar is an activated nucleotide sugar, for example ADP-glucose or UDP-glucose. The present invention also provides a method of preparing DNA, wherein the DNA has at least a proportion of cytosine residues as glucosyl hydroxymethylcytosine (ghmC) residues, comprising contacting isolated DNA having at least a proportion of cytosine residues as hydroxymethylcytosine (hmC) with a glucosyl transferase in the presence of a sugar. It will be appreciated that a number of sugars may be used, for example an activated nucleotide sugar or galactose, mannose, acetyl glucosamine, fucose, xylose, rhamnose or fructose. Usually, the sugar will be an activated nucleotide sugar, for example ADP-glucose, UDP-glucose, UDP-a-D-GIc, UDP-a-D- Gal, UDP-a-D-GalNAc, UDP-a-D-GlcNAc, UDP-a-D-GIcA, UDP-a-D-Xyl, GDP-a-D- Man, GDP- -L-Fuc, CMP- -D-Neu5Ac, CDP-glucose and TDP-glucose. Preferably the sugar is an activated nucleotide sugar, for example ADP-glucose or UDP- glucose.

The present invention also provides a method of transforming a prokaryotic cell with a desired DNA molecule other than by a T-even phage, comprising introducing the desired DNA into the cell, wherein at least a proportion of the cytosine residues of the DNA are glycosyl hydroxymethylcytosine residues, and wherein the said prokaryotic cell is not transformable with the same desired DNA molecule lacking glycosyl hydroxymethylcytosine residues. Preferably, the cell modified by the method is a bacterial cell, preferably an E. coli cell. Preferably at least a proportion of the cytosine residues of the DNA are glucosyl hydroxymethylcytosine (ghmC) residues.

Preferably, the desired DNA having at least a proportion of the cytosine residues of the DNA as glycosyl hydroxymethylcytosine residues is comprised in a plasmid produced by a prokaryotic cell of the invention.

Advantageously, the present invention also provides a method of genetically modifying a cell by a process involving homologous recombination, comprising creating a double stranded break at a desired locus and introducing a donor template DNA molecule into the cell, wherein the template DNA has at least a proportion of cytosine residues as hydroxymethylcytosine (hmC) or glycosyl hydroxymethylcytosine residues. Preferably the template DNA is impervious to degradation by sequence-specific nucleases, for example Cas9. Preferably, the double stranded DNA break is created by a nuclease. A number of nucleases will be suitable for this purpose. Preferably such a nuclease will be a Cas3 nuclease or a Cas9 nuclease. Preferably the nuclease will be a Cas9 nuclease complexed with a guide RNA (gRNA), a portion of which having sequence complementarity to the target sequence.

Modification of the cytosines of the repair template in this way allows the repair template to evade sequence-specific degradation by the cell, either by the native nucleases of the cell or by a CRISPR-Cas construct which is expressed in the cell to mediate the modification of the cellular DNA. This improves the efficiency of genetic engineering of cellular DNA and allows editing of sequences close to PAM sites.

Preferably, the template DNA has at least one hydroxymethylcytosine (hmC) or glycosyl hydroxymethylcytosine residue in the PAM region and/or protospacer region. Preferably the template DNA is impervious to degradation by sequence- specific nucleases, for example Cas9. Consequently, the PAM region may be retained in the repair template and thus high-levels of homology with sequence regions immediately upstream and/or downstream of the intended editing site, which improves the efficiency of HDR in comparison to sequences in which the PAM sequence is excluded or mutated to avoid sequence-specific degradation of the repair template prior to or after genome repair by the Cas9 nuclease-guideRNA complex. Preferably, the template DNA has at least a proportion of cytosine residues as glucosyl hydroxymethylcytosine (ghmC) residues. Preferably, at least one glucosyl hydroxymethylcytosine (ghmC) residue is located in the PAM region and/or protospacer region of the DNA repair template. The cell modified by the method may be a prokaryotic or a eukaryotic cell. Preferably, the cell modified by the method is a prokaryotic cell. More preferably, the cell modified by the method is a bacterial cell, preferably an E. coli cell. The present invention also provides a method of genetically modifying an organism by a process involving homologous recombination, comprising creating a double stranded DNA break at a desired locus using a Cas9 nuclease, and introducing a donor template DNA molecule in the cell, wherein the template DNA has at least one modified cytosine residue in the PAM region and/or protospacer region, to prevent cleavage of the template DNA before or after genome integration.

According to methods of the invention, transformation of a cell with a desired DNA molecule is preferably achieved by direct transformation in the presence of the transforming polynucleotides or expression constructs. Suitable methods of direct transformation include electroporation, polyethylene glycol (PEG) treatment, heat- shock, high pH treatment, microinjection, electrophoresis, silicon carbide- or liposome-mediated transformation, lipid-mediated transfection or calcium phosphate transfection or by viral delivery, for example lentivirus, Adenovirus, AAV. However, it will be appreciated that cells may be transformed with a desired polynucleotide by a number of other techniques, if desired. Alternatively in the case of plant cells, or protoplasts, gene transfer via a disarmed Ti-plasmid vector carried by Agrobacterium tumefaciens, using Agrobacterium sp. -mediated transformation or vacuum infiltration may also be used. Alternatively, a combination of different techniques may be employed to enhance the efficiency of the transformation process depending on the desired application.

BRIEF DESCRIPTION OF THE FIGURES

The invention will now be described in detail with reference to examples and with reference to the accompanying drawings, in which:

Figure 1 shows a schematic comparison of the structures of Cytosine, hydroxymethylcytosine (hmC) and glucosyl hydroxymethylcytosine (ghmC). Figure 2 shows a diagram of the plasmid pMK-RQ containing the ghmC operon (56, β-gt, 1 , 42) comprising the polynucleotide sequences encoding deoxycytidylate 5- hydroxymethyltransferase (dCMP Hmase) (42), deoxy-Nucleotide Monophosphate (dNMP) kinase (1 ), deoxycytidine-triphosphatase (dCTPase) (56) and β-glucosyl transferase (β-gt). Please note that the terminator upstream of the dCTPase gene is optional and can be removed to enhance expression of the dCTPase gene.

Figure 3 shows a flow chart illustrating the production of unmodified DNA in E. coli, (panel A) and ghmC DNA in T4 phage (panel B) and ghmC DNA in E. cloni pGhmC (panel C).

Figure 4 shows growth (OD600) of cultures producing GGO (diamonds), hmC (triangles) and ghmC (squares) over time (hours). GGO refers to a control plasmid comprising an identical plasmid backbone with an insert unrelated to DNA modifications.

Figure 5 shows a gel picture of restriction of DNA by modification sensitive and/or specific enzymes. Lanes 1 , 4 and 7 show no restriction enzyme controls. Lanes 2, 5 and 8 show cleavage with AbaSI. Lanes 3, 6 and 9 show cleavage with McrBC. Abbreviations are as follows: Lanes 1 -3 pGhmC: plasmid containing four T4 genes involved in GhmC formation (Gene 56: dCTPase; Gene 42: dCMP hydroxymethylase; Gene 1 : dNMP kinase; Gene β-gt: beta-glucosyltransferase). Lanes 3-6 pHmC: plasmid containing three T4 genes involved in 5-hmC formation (Gene 56: dCTPase; Gene 42: dCMP hydroxymethylase; Gene 1 : dNMP kinase). Lanes 3-9 pGGO (empty plasmid control plasmid).

Below are polynucleotide and amino acid sequences of genes and proteins used in accordance with the invention.

SEQ ID NO: 1 Amino Acid sequence of deoxycvtidylate 5- hydroxymethyltransferase (dCMP Hmase) (Gene 42)

MISDSMTVEEIRLHLGLALKEKDFVVDKTGVKTIEIIGASFVADEPFIFGALNDEYIQR ELEWYKSKSLFVKDIPGETPKIWQQVASSKGEINSNYGWAIWSEDNYAQYDMCLA ELGQNPDSRRGIMIYTRPSMQFDYNKDGMSDFMCTNTVQYLIRDKKINAWNMRS NDWFGFRNDYAWQKYVLDKLVSDLNAGDSTRQYKAGSIIWNVGSLHVYSRHFYL VDHWWKTGETH ISKKDYVGKYA SEQ ID NO: 2 Coding sequence of deoxycytidylate 5- hydroxymethyltransferase (dCMP Hmase) (Gene 42)

TTAAGCGTATTTTCCTACATAATCTTTTTTCGAAATATGTGTTTCGCCAGTTTTCC ACCAATGATCAACCAAATAAAAATGACGAGAATACAatCATGTAGGCTTCCAACA TTCCATATAATGGAACCTGCTTTATACTGGCGAGTTGAATCACCTGCATTCAAAT CAGATACTAATTTATCTAATACGTATTTTTGCCATGCATAATCATTACGGAATCC GAAGACCACGTCATTTGAGCGCATGTTAACAACCGCATTGATTTTCTTGTCACG AATCAGGTATTGTACTGTATTCGTGCACATGAAATCTGACATACCATCTTTATTA TAGTCAAACTGCATAGATGGACGAGTATAAATCATGATACCACGTCGAGAATCA GGATTTTGACCAAGTTCAGCTAAACACATGTCATACTGAGCATAGTTATCTTCTG ACCAGATAGCCCAACCATAATTCGAGTTAATTTCACCTTTAGAAGATGCTACTTG TTGCCAAATCTTCGGTGTTTCACCCGGAATATCTTTAACGAACAAGCTTTTAGAT TTATACCATTCAAGTTCACGCTGAATGTATTCATCATTAAGAGCGCCAAAAATAA ACGGTTCATCTGCTACAAATGATGCGCCAATAATTTCAATAGTTTTAACACCTGT TTTATCAACTACGAAATCTTTTTCTTTTAATGCAAGCCCCAAATGAAGACGGATT TCTTCAACTGTCATAGAGTCACTAATCAT

SEQ ID NO: 3 Amino acid sequence of deoxy-Nucleotide Monophosphate (dNMP) kinase (Gene 1)

MKLIFLSGVKRSGKDTTADFIMSNYSAVKYQLAGPIKDALAYAWGVFAANTDYPCLT RKEFEGIDYDRETNLNLTKLEVITIMEQAFCYLNGKSPIKGVFVFDDEGKESVNFVAF NKITDVINNIEDQWSVRRLMQALGTDLIVNNFDRMYWVKLFALDYLDKFNSGYDYYI VPDTRQDHEMDAARAMGATVIHVVRPGQKSNDTHITEAGLPIRDGDLVITNDGSLE ELFSKIKNTLKVL

SEQ ID NO: 4 Coding sequence of deoxy-Nucleotide Monophosphate (dNMP) kinase (Gene 1)

TTATAGTACCTTTAGTGTATTTTTAATTTTAGAAAAAAGTTCTTCAAGAGAACCAT CGTTTGTAATTACTAAATCGCCATCACGAATTGGCAATCCAGCTTCTGTAATATG TGTATCATTGGATTTTTGACCAGGACGAACTACATGAATTACTGTAGCACCCATC GCCCTAGCCGCATCCATTTCATGATCTTGACGGGTATCAGGAACGATATAATAA TCATAACCTGAGTTAAATTTATCAAGATAATCTAAAGCAAATAATTTTACCCAGTA CATGCGGTCGAAGTTATTAACAATCAAATCCGTACCTAGGGCTTGCATCAGACG ACGGACTGACCATTGATCTTCAATATTATTTATAACGTCAGTAATCTTATTAAATG CTACGAAATTAACTGATTCTTTTCCTTCGTCATCAAAAACAAACACACCTTTAATT GGGCTTTTACCATTAAGATAACAAAATGCTTGTTCCATAATCGTGATTACTTCTA ATTTAGTCAGATTTAAATTAGTCTCACGATCATAGTCAATTCCTTCAAACTCTTTA CGAGTTAAGCAAGGATAGTCAGTGTTTGCTGCAAATACTCCCCATGCATAAGCC AATGCATCCTTAATGGGACCAGCAAGTTGGTATTTAACTGCAGAATAATTGCTCA TGATAAAATCAGCAGTAGTATCTTTTCCACTACGCTTTACACCGCTTAAAAAGAT TAGTTTCAT

SEQ ID NO: 5 Amino acid sequence of dCTPase (Gene 56)

MAHFNECAHLIEGVDKAQNEYWDILGDEKDPLQVMLDMQRFLQIRLANVREYCYH PDKLETAGDVVSWMREQKDCIDDEFRELLTSLGEMSRGEKEASAVWKKWKARYIE AQEKRIDEMSPEDQLEIKFELVDIFHFVLNMFVGLGMNAEEIFKLYYLKNKHNFERQ DNGY SEQ ID NO: 6 Coding sequence of dCTPase (Gene 56)

ATGGCTCACTTTAATGAATGTGCTCATTTGATCGAAGGTGTTGATAAAGCTCAAA ATGAATACTGGGATATTCTCGGTGATGAAAAAGATCCGCTGCAAGTTATGCTTG ATATGCAGCGGTTTTTACAGATTCGTTTGGCTAATGTCCGCGAATACTGCTATCA TCCAGATAAATTAGAAACTGCCGGTGATGTTGTTTCTTGGATGCGTGAACAAAA AGACTGTATTGATGATGAATTTCGCGAACTTCTGACTTCTCTTGGTGAAATGTCA CGTGGTGAAAAAGAAGCTTCTGCTGTATGGAAAAAATGGAAAGCACGTTATATT GAAGCGCAAGAAAAACGCATTGATGAAATGTCCCCCGAAGACCAGCTCGAAAT TAAATTTGAGCTTGTGGATATATTTCATTTCGTATTAAATATGTTTGTTGGCCTTG GAATGAATGCGGAAGAAATCTTTAAACTTTATTATCTGAAGAACAAACATAATTTT GAACGTCAAGATAATGGATATTAA

SEQ ID NO: 7 Amino acid sequence of beta-qlucosyltransf erase (β-gt)

MKIAIINMGNNVINFKTVPSSETIYLFKVISEMGLNVDIISLKNGVYTKSFDEVDVNDY DRLIWNSSINFFGGKPNLAILSAQKFMAKYKSKIYYLFTDIRLPFSQSWPNVKNRPW AYLYTEEELLIKSPIKVISQGINLDIAKAAHKKVDNVIEFEYFPIEQYKIHMNDFQLSKP TKKTLDVIYGGSFRSGQRESKMVEFLFDTGLNIEFFGNAREKQFKNPKYPWTKAPV FTGKIPMNMVSEKNSQAIAALIIGDKNYNDNFITLRVWETMASDAVMLIDEEFDTKH RIINDARFYVNNRAELIDRVNELKHSDVLRKEMLSIQHDILNKTRAKKAEWQDAFKK AIDL SEQ ID NO: 8 Coding sequence of beta-qlucosyltransf erase NT (β-gt)

TTATAAATCAATAGCTTTTTTGAACGCATCTTGCCATTCGGCTTTCTTTGCACGG GTTTTATTTAAAATATCATGTTGAATAGAAAGCATCTCTTTACGCAAAACATCACT GTGTTTTAACTCATTGACTCTATCAATGAGTTCAGCACGATTATTTACATAAAAAC GAGCATCATTAATAATTCGATGTTTGGTATCAAATTCTTCGTCAATTAGCATCAC TGCATCAGATGCCATTGTTTCCCAGACACGTAAGGTAATAAAGTTGTCATTATAA TTCTTGTCACCAATAATTAATGCAGCAATAGCTTGACTATTCTTTTCAGATACCAT GTTCATAGGAATTTTTCCAGTGAACACCGGAGCTTTTGTCCAAGGATATTTAGG ATTTTTAAACTGTTTTTCTCGTGCATTGCCAAAAAACTCAATATTTAAACCGGTGT CAAATAAAAATTCTACCATCTTGGATTCGCGTTGACCAGACCGAAATGAACCGC CATAAATAACATCCAAAGTTTTCTTGGTAGGCTTAGATAATTGAAAATCGTTCAT ATGAATTTTATATTGTTCAATAGGAAAATATTCAAATTCAATAACATTATCAACTTT CTTATGCGCAGCCTTAGCAATGTCTAAATTTATACCTTGGGAAATCACTTTAATT GGTGATTTGATTAATAGCTCTTCTTCAGTGTACAAATATGCCCAAGGTCTATTTT TAACATTTGGCCAAGACTGCGAAAACGGCAAACGTATATCTGTAAATAAATAATA AATTTTACTTTTGTATTTTGCCATAAATTTTTGCGCAGATAAAATTGCTAAATTAG GTTTACCGCCAAAAAAGTTAATAGAAGAATTAACAACTATCAAACGGTCATAATC ATTAACATCTACTTCATCAAAAGATTTAGTGTAAACACCATTTTTAAGAGAAATAA TATCGACATTAAGACCCATTTCAGAAATAACTTTAAAAAGATAAATAGTTTCAGAA GATGGAACAGTTTTAAAATTAATAACATTATTACCCATATTAATTATAGCAATTTT CAT

SEQ ID NO: 9 polynucleotide sequence of ghrnC operon (genes 56, B-qt, 1, 42 (appearing in this order in underlined italic text)) correspond also to SEQ ID NOs: 1 , 3, 5 and 7 respectively in plasmid pMK-RQ backbone

>Operon_in_pMK-RQ Confirmed by sequencing

CTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCA GCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGA ATAGACCGAGATAGGGTTGAGTGGCCGCTACAGGGCGCTCCCATTCGCCATTC AGGCTGCGCAACTGTTGGGAAGGGCGTTTCGGTGCGGGCCTCTTCGCTATTAC GCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCC AGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGACGTAA TACGACTCACTATAGGGCGAATTGAAGGAAGGCCGTCAAGGCCGCATATGCAT GCATGAATTCGATGCATCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGA CTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCAC ACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATAGAATTCAACCATGGAAG ATCTTTTAAG AAG G AG ATATAC A^"17\ TGGCTCACTTTAA TGAA TGTGCTCA TTTGA TCGAAGGTGTTGATAAAGCTCAAAATGAATACTGGGATATTCTCGGTGATGAAA AA GA TCCGCTGCAA G TTA TGCTTGA TA TGCAGCGGTTTTTA CA GA TTCGTTTGG CTAA TGTCCGCGAA TACTGCTA TCA TCCA GA TAAA TTAGAAACTGCCGGTGA TG TTGTTTCTTGGATGCGTGAACAAAAAGACTGTATTGATGATGAATTTCGCGAACT TCTGACTTCTCTTGGTGAAATGTCACGTGGTGAAAAAGAAGCTTCTGCTGTATG GAAAAAA TGGAAAGCACGTTA TA TTGAAGCGCAA GAAAAACGCA TTGA TGAAA T G TCCCCCGAA GA CCAGCTCGAAA TTAAA TTTGAGCTTG TGGA TA TA TTTCA TTTC GTA TTAAATA TGTTTGTTGGCCTTGGAATGAA TGCGGAAGAAA TCTTTAAACTTT A TTA TCTGAA GAACAAACA TAA TTTTGAACGTCAAGA TAA TGGA TA TTAACAATT GGTCGACAAATAATAAAAAAGCCGGATTAATAATCTGGCTTTTTATATTCTCTCG TACGCGATCG TTA TAAA TCAA TAGCTTTTTTGAACGCA TCTTGCCA TTCGGCTTT CTTTGCACGGGTTTTA TTTAAAA TA TCA TGTTGAATAGAAAGCATCTCTTTACGC AAAACATCACTGTGTTTTAACTCATTGACTCTATCAATGAGTTCAGCACGATTAT TTACA TAAAAACGA GCA TCA TTAA TAA TTCGA TG TTTGGTA TCAAA TTCTTCGTCA ATTAGCATCACTGCATCAGATGCCATTGTTTCCCAGACACGTAAGGTAATAAAG TTG TCA TTA TAA TTC TTG TCA CCAA TAA TTAA TG CA GCAA TAGC TTGA C TA TTC TT TTCA GA TA CCA TG TTCA TA G GAA TTTTTCCA G TGAA CACCGGAGC TTTTG TCCAA GGA TA TTTAGGA TTTTTAAACTGTTTTTCTCGTGCA TTGCCAAAAAACTCAA TA TT TAAACCGGTGTCAAATAAAAATTCTACCATCTTGGATTCGCGTTGACCAGACCG AAATGAACCGCCATAAATAACATCCAAAGTTTTCTTGGTAGGCTTAGATAATTGA AAA TCGTTCA TA TGAA TTTTA TA TTGTTCAA TAGGAAAA TA TTCAAA TTCAA TAAC A TTA TCAACTTTCTTA TGCGCAGCCTTA GCAA TGTCTAAA TTTA TACCTTGGGAA ATCACTTTAATTGGTGATTTGATTAATAGCTCTTCTTCAGTGTACAAATATGCCCA AGGTCTATTTTTAACATTTGGCCAAGACTGCGAAAACGGCAAACGTATATCTGTA AA TAAA TAA TAAA TTTTACTTTTG TA TTTTGCCA TAAA TTTTTGCGCA GA TAAAA TT GCTAAATTAGGTTTACCGCCAAAAAAGTTAATAGAAGAATTAACAACTATCAAAC GGTCATAATCATTAACATCTACTTCATCAAAAGATTTAGTGTAAACACCATTTTTA A GA GAAA TAA TA TC GA CA TTAA GACCCA TTTCA GAAA TAA C TTTAAAAA GA TAAA TA G TTTCA GAA GA TG GAA CA G TTTTA AAA TTAA TAA CA TTA TTACCCA TA TTAA TT A TAGCAA TTTTCA 7ATGTATATCTCCTTCTTTAATTAAGGCGCGCCTTATAGTACC TTTAGTGTATTTTTAATTTTAG AAAAAAGTTCTTCAAG AG AACCATC G TTTG TAA T TACTAAA TCGCCATCACGAA TTGGCAA TCCAGCTTCTGTAA TA TGTGTA TCA TTG GA TTTTTGACCAGGACGAACTACA TGAA TTACTGTA GCACCCA TCGCCCTAGCC GCA TCCA TTTCA TGA TCTTGACGGGTA TCAGGAACGA TA TAA TAA TCA TAACCTG A G TTAAA TTTA TCA A GA TAA TC TAAA G CAAA TAA TTTTA CCCAGTACATGCGGTC GAAGTTATTAACAATCAAATCCGTACCTAGGGCTTGCATCAGACGACGGACTGA CCA TTGA TCTTCAA TA TTA TTTA TAACGTCAGTAA TCTTA TTAAA TGCTACGAAA T TAACTGATTCTTTTCCTTCGTCATCAAAAACAAACACACCTTTAATTGGGCTTTTA CCA TTAA GA TAA CAAAA TG C TTG TTCCA TAA TC G TGA TTA C TTC TAA TTTA G TCA G ATTTAAATTAGTCTCACGATCATAGTCAATTCCTTCAAACTCTTTACGAGTTAAGC AAGGA TA G TCA G TG TTTGCTGCAAA TACTCCCCA TGCA TAAGCCAA TGCA TCCT TAATGGGACCAGCAAGTTGGTA TTTAACTGCAGAA TAA TTGCTCA TGA TAAAA TC AGCAGTAGTATCTTTTCCACTACGCTTTACACCGCTTAAAAAGATTAGTTTCATk TGTkTkTCTCCTTCTkCGCGTCCCGGGTTAAGCGTATTTTCCTACATAATCTTTT TTCGAAA TA TG TG TTTCGCCA G TTTTCCA CCAA TGA TCAACCAAA TAAAAA TGA C GA GAA TA CAatCA TG TA GGCTTCCAA CA TTCCA TA TAA TG GAACCTGCTTTA TACT GGCGAGTTGAATCACCTGCATTCAAATCAGATACTAATTTATCTAATACGTATTT TTGCCA TGCA TAA TCA TTACGGAA TCCGAA GA CCACGTCA TTTGAGCGCA TG TT AA CAA CCGCA TTGA TTTTCTTG TCACGAA TCA GGTA TTG TACTG TA TTCGTGCA C ATGAAATCTGACATACCATCTTTATTATAGTCAAACTGCATAGATGGACGAGTAT AAATCATGATACCACGTCGAGAATCAGGATTTTGACCAAGTTCAGCTAAACACA TGTCATACTGAGCATAGTTATCTTCTGACCAGATAGCCCAACCATAATTCGAGTT AA TTTCACCTTTAGAAGA TGCTACTTGTTGCCAAA TCTTCGGTGTTTCACCCGGA ATATCTTTAACGAACAAGCTTTTAGATTTATACCATTCAAGTTCACGCTGAATGTA TTCA TCA TTAA GAGCGCCAAAAA TAAACGGTTCA TCTGCTACAAA TGA TGCGCC AATAATTTCAATAGTTTTAACACCTGTTTTATCAACTACGAAATCTTTTTCTTTTAA TGCAAGCCCCAAATGAAGACGGATTTCTTCAACTGTCATAGAGTCACTAATCAT ATGTATATCTCCTTCTTCCGGAGAACTTTTCAATTCGCGTTAAACAAAATTATTAC TAGAGGGAAACCGCCAGGGTCTCCCTACGACCAGTCTAAAAAGCGCCTCAATT CGCGACCTTCTCGTTACTGACAGGAAAATGGGCCATTGGCAACCAGGGAAAGA TGAACGTGATGATGTTCACAATTTGCTGAATTGTGGTGGACGAATTTGGATCCC TGGGCCTCATGGGCCTTCCTTTCACTGCCCGCTTTCCAGTCGGGAAACCTGTC GTGCCAGCTGCATTAACATGGTCATAGCTGTTTCCTTGCGTATTGGGCGCTCTC CGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGGTAAAGCCTGGGG TGCCTAATGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGT TGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGA CGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTT TCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCG GATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCAC GCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTG CACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCT TGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTA ACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGG TGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTG AAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACC ACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAA AAAG G ATCTCAAG AAG ATCCTTTG ATCTTTTCTACG G G GTCTG ACGCTCAGTG G AACG AAAACTCACGTTAAG G G ATTTTG GTCATGAG ATTATCAAAAAG G ATCTTCA CCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAG TAAACTTGGTCTGACAGTTATTAGAAAAATTCATCCAGCAGACGATAAAACGCAA TACGCTGGCTATCCGGTGCCGCAATGCCATACAGCACCAGAAAACGATCCGCC CATTCGCCGCCCAGTTCTTCCGCAATATCACGGGTGGCCAGCGCAATATCCTG ATAACGATCCGCCACGCCCAGACGGCCGCAATCAATAAAGCCGCTAAAACGGC CATTTTCCACCATAATGTTCGGCAGGCACGCATCACCATGGGTCACCACCAGAT CTTCGCCATCCGGCATGCTCGCTTTCAGACGCGCAAACAGCTCTGCCGGTGCC AGGCCCTGATGTTCTTCATCCAGATCATCCTGATCCACCAGGCCCGCTTCCATA CGGGTACGCGCACGTTCAATACGATGTTTCGCCTGATGATCAAACGGACAGGT CGCCGGGTCCAGGGTATGCAGACGACGCATGGCATCCGCCATAATGCTCACTT TTTCTGCCGGCGCCAGATGGCTAGACAGCAGATCCTGACCCGGCACTTCGCCC AGCAGCAGCCAATCACGGCCCGCTTCGGTCACCACATCCAGCACCGCCGCAC ACGGAACACCGGTGGTGGCCAGCCAGCTCAGACGCGCCGCTTCATCCTGCAG CTCGTTCAGCGCACCGCTCAGATCGGTTTTCACAAACAGCACCGGACGACCCT GCGCGCTCAGACGAAACACCGCCGCATCAGAGCAGCCAATGGTCTGCTGCGC CCAATCATAGCCAAACAGACGTTCCACCCACGCTGCCGGGCTACCCGCATGCA GGCCATCCTGTTCAATCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAG GGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAA TAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCAC DETAILED DESCRIPTION

Example 1 : Synthesis of ghmC DNA

Biosynthesis of DNA precursors in E. coli can be divided into two pathways, the pyrimidine and purine metabolism pathways. The pyrimidine pathway leads to the production of dTTP and dCTP, the purine metabolism pathway leads to the production of dGTP and dATP. A set of phage encoded enzymes is responsible for the production of hmdCTP that is incorporated into the newly synthesized DNA. Also these enzymes exclude the incorporation of dCTP into DNA. Phage T4 encodes several proteins that, together with two host proteins, form the dNTP synthase complex (DSC). This complex is a very efficient complex that breaks down the deoxycytidine triphosphate (dCTP) pool and generates hydroxymethyl-deoxycytidine triphosphate (hmdCTP). The DSC consists of at least 10 proteins, 8 phage encoded and 2 host encoded (Table 2). Table 2. Enzymes of the dNTP synthase complex (DSC).

Example 2: Expression of ghmC operon in E. coli

In an effort to produce hmC and its glucosylated derivate ghmC a plasmid was constructed encoding the minimal genes required for the production of ghmC; gene 42, 1 , 56, and B-gt, encoding dCMP hydroxymethylase, dNMP kinase, dCTPase, and B-glucosyltransferase respectively. Genes 42, 1 , and 56 are under the control of a rhamnose inducible promoter. Gene 56 is under the control of an IPTG inducible T7 promoter. Cultures were induced and plasmid DNA was isolated followed by restriction with hmC/ghmC sensitive or specific enzymes. Incubation with AbaSI Cleavage patterns of AbaSI and X indicate that the DNA is partly (glucosyl)-hydroxymethylated.

Example 3: Degree of substitution

To investigate which of these four genes are essential for the production of hmC/ghmC, plasmids were constructed lacking either gene 42, 1 , 56, or β-gt. Restriction digest analysis shows that all four genes are essential for the production of ghmC (as shown in Table 3). As expected, the deletion of gene β-gt results in the production of hmC DNA. The deletion of gene 1 results in C-DNA, showing that the dNMP kinase is essential. The deletion of gene 56 results in the production of ghmC DNA but the degree of base substitution is lower.

Table 3. Selectivity of restriction enzymes on modified DNA.

Enzyme C hmC ghmC

Ndel Y Y Y

CviQI Y Y X

McrBC X Y X

AbaSI X w Y

Hindlll Y X X

MspJI N Y N Example 4: Stability of the system

When cells containing pGhmC were cultured for a period longer than one day, isolated DNA would not be cleaved by AbaSI. This indicated some instability of the expression of the construct. To further investigate the toxicity of the expression of the ghmC operon growth curve experiments were performed. Cells containing pGhmC or pHmC showed a longer lag-phase, slower growth in the exponential phase, and a lower cell density in the stationary phase compared with cells containing the control plasmid pGGO.

When cells were grown in the absence of inducer, a relatively high expression of pGhmC or pHmC was observed (SDS-PAGE analysis). To achieve the greatest degree of cytosine substitution, cells were grown overnight in the presence of glucose to repress expression of genes under control of the Rhamnose promoter. Overnight cultures (OD600 of around 2) were used to inoculate fresh LB medium containing 0.2% Rhamnose as inducer. Restriction of isolated plasmid and genomic DNA showed an increased degree of cytosine substitution than DNA isolated from cultures that were induced at the inoculation point.

Example 5: Deglucosylation of ghmC DNA

As shown above, expression of the hmC operon enables to produce hmC DNA in E. coli and expression of B-glucosyltransferase results in the glucosylated hmC DNA. To understand and control the cytosine substitution system in more depth the glucosyl groups were removed from ghmC DNA by using B-glucosidase. ghmC DNA was incubated with b-glucosidase and freed glucose was measured using a glucose detection kit.

Example 6: Phages propagated in E. cloni pGhmC contain ghmC DNA

The improved biological stability of ghmC can be exploited to protect bacteriophage or host DNA in view of the ever-ongoing arms race between phages and their prokaryotic hosts. Expression of the ghmC operon in E. coli affects the DNA synthesis in the entire cell, thus also replication of phage DNA upon infection. To test our hypothesis that phages propagated in E. cloni containing pGhmC would also contain ghmC DNA phage infection assays were carried out. A virulent strain of phage λ, (A_Vi_r), was propagated in E. coli containing pGhmC and used to infect E. coli.

Example 7: Availability of UDP-glucose in E. coli

UDP-glucose in E. coli is used as substrate for the synthesis of other UDP-sugars such as UDP-galactose. It is formed by the reaction of UTP with glucose-1 - phosphate catalysed by UDP-glucose pyrophosphorylase (GalU). Even though the genotype of E. cloni 10G shows that it is galU^", it does produce UDP-glucose. E. cloni 10G contains a frame shift mutation in the second codon of the galU gene resulting in an early stop codon. 65bp downstream the start codon is another ATG start codon located. The large ORF most likely this results in a smaller, but still functional, UDP-glucose pyrophosphorylase.

Example 8: General Materials and Methods i) Strains and constructs

E. cloni 10G (Lucigen) (relevant genotype: endA1 recA1 mcrA A(mrr-hsdRMS- mcrBC) galU galK) was used for all analytical experiments. E. cloni 10G was made chemically competent using the RuCI method and transformed with pGhmC by applying a heat-shock as described in the QIAexpressionist handbook (QIAGEN). Cells were grown in Luria Broth (LB; 10 g-L-1 NaCI, 5 g-L-1yeast extract, and 10 g-L-1 tryptone) at 180 rpm or on LB-agar plates containing 1 .5% (wt-vol-1 ) agar. To repress expression of the construct, 0.2% glucose was supplemented to the medium. 2% Rhamnose was used for induction of protein expression. When required, medium was supplemented with kanamycin (Km; 50 pg-mL-l ). Bacterial growth was measured at 600 nm (OD600). Sequences of genes 42, 1 , β-gt, and 56 were selected from phage T4 (GenBank: AF158101 .6) and unwanted restriction sites were deleted. The operon was assembled from synthetic oligonucleotides and/or PCR products and cloned into pMK-RQ (kanR) by GeneArt (Life Technologies). E. coli K12 was transformed with the synthetic construct and cells were grown followed by plasmid isolation. All subsequent experiments were performed in E. cloni 10G.

To create the constructs that lack one of the ghmC operon genes, pGhmC was restricted using the enzymes listed in Table 4. Following restriction, plasmids were re-ligated using T4 DNA ligase and used to transform E. cloni 10G. The control plasmid pFS containing a frame-shift and thus non-functional mRNA production, was produced by performing a PCR using primers BG6559 and 6560 followed by ligation and transformation. Table 4. Enzymes used for construction of plasmids lacking one gene of the ghmC operon.

ii) Molecular Biology and DNA Sequencing

All oligonucleotides are listed in Table 5. All strains and plasmids were confirmed by PCR and sequencing (GATC-Biotech). Plasmids were prepared using GeneJET Plasmid Miniprep Kits (Thermo Scientific) and DNA from PCR and agarose gels was purified using the Thermo Scientific GeneJET PCR Purification and Gel Extraction Kits. The bacteriophages that are used herein are phage T4 and T4gt. Phage T4wt (CBS-KNAW Biodiversity Centre) was propagated in E. coli B834 (Su-) and has glucosylated 5-hydroxymethylcytosines (ghmC) in its DNA. Phage T4gt (a gift from NEB) (relevant genotype: a-gt57 and β-gtl 4) is deficient in both a- and β- glucosyltransferase and was propagated in E. cloni (Georgopoulos, 1967). Phage DNA was extracted by using the phenol chloroform isoamylalcohol (PCI) method.

Table 5. Primers used in this study

BG6622 ACGGCGTCGATTGCTCAACGC galU seq REV

BG6734 GCGCGCGAATTCAACCATGGAAGA GhmC operon FW EcoRI for

TC 1 1 1 1 AAGAAG lambda gt1 1

BG6735 GCGCGCGAATTCCAGGGATCCAAA Ghmc operon REV EcoRI for

TTC lambda gt1 1

iii) Restriction analysis of DNA

DNA was restricted using 10U of enzyme in the recommended buffers and conditions. The products were visualized on a 1 % agarose gel that was stained after running the gel.

iv) SDS-PAGE

Cells containing pGhmC or pGGO (control) were cultured overnight in the presence of 0.2% glucose to repress expression of the GhmC operon. The cells were re- inoculated in medium containing 0.2% Rhamnose to induce expression of the GhmC operon. Cells were harvested and lysed using sonication. Crude cell lysates were analysed in a 10% SDS-PAGE gel using PAGE-Blue protein stain. Molecular sizes of proteins were compared to a precision plus (BioRad) protein standard.

Example 9: AbaSI cleaves ghmC DNA very well, and hmC DNA poorly, and does not cleave normal DNA; McrBC cleaves only hmC DNA

Figure 5 shows the results of restriction of DNA by modification sensitive and/or specific enzymes. pGhmC, pHmC and pGGO (control plasmid) were all tested for cleavage with AbaSI and McrBC and compared with no restriction enzyme controls. AbaSI is a DNA modification-dependent endonuclease which recognizes 5- glucosylhydroxymethylcytosine (5ghmC) in double stranded DNA. It also recognizes 5-hydroxymethylcytosine (5hmC) but at a lower efficiency. AbaSI does not recognize DNA with 5-methylcytosine or un-modified cytosine. AbaSI selectively cleaves DNA that contains the modified bases, 5ghmC or 5hmC on one or both strands and introduces a double-stranded DNA break on the 3^' side away from the modified cytosine producing a 2-base or 3-base 3'-overhang. Sites with two 5ghmC on opposite strands are cleaved most efficiently; sites with one 5ghmC and another C or 5mC are cleaved less efficiently.

McrBC is an endonuclease which cleaves DNA containing methylcytosine on one or both strands. McrBC does not act upon unmethylated DNA. pGhmC: plasmid containing four T4 genes involved in GhmC formation

· Gene 56: dCTPase, degrades the host dCTP pool, blocking normal DNA

production

• Gene 42: dCMP hydroxymethylase, creates HmdCMP from dCMP

• Gene 1 : dNMP kinase, phosphorylates HmdCMP to HmdCDP

• Gene β-gt: beta-glucosyltransferase, glucosylates hmC-DNA pHmC: plasmid containing three T4 genes involved in 5-hmC formation

• Gene 56: dCTPase, degrades the host dCTP pool, blocking normal DNA

production

• Gene 42: dCMP hydroxymethylase, creates HmdCMP from dCMP

• Gene 1 : dNMP kinase, phosphorylates HmdCMP to HmdCDP pGGO: empty plasmid control plasmid, comprising an identical plasmid backbone with an insert unrelated to DNA modifications.

AbaSI cleaves ghmC DNA very well, and hmC DNA poorly, and does not cleave normal DNA.

McrBC cleaves only hmC DNA.

Claims

1 . A genetically engineered cell, free of T-even phage infection, comprising a

deoxycytidylate 5-hydroxymethyltransferase (dCMP Hmase) gene and a deoxy- Nucleotide Monophosphate (dNMP) kinase gene.

2. A cell as claimed in claim 1 , further comprising a deoxycytidine-triphosphatase (dCTPase) gene.

3. A cell as claimed in claim 1 or claim 2, further comprising a glycosyl transferase gene.

4. A cell as claimed in claim 3, wherein the glycosyl transferase gene is a glucosyl transferase gene, preferably an a and/or β glucosyl transferase gene.

5. A cell as claimed in any of claims 3 or 4, wherein substantially all of the cytosine residues of the DNA of the cell are glycosyl hydroxymethylcytosine residues.

6. A cell as claimed in claim 5, wherein substantially all of the cytosine residues of the DNA of the cell are glucosyl hydroxymethylcytosine (ghmC) residues.

7. A cell as claimed in any preceding claim, wherein the cell is a prokaryotic cell.

8. A cell as claimed in claim 7, wherein the genes are comprised in a plasmid.

9. A cell as claimed in claim 7 or claim 8, wherein expression of the genes is under operative control of a (single) promoter, preferably an inducible promoter for example a Rhamnose inducible promoter or a trc promoter.

10. A cell as claimed in any of claims 7 to 9, wherein expression of the genes is under the control of a Rhamnose inducible promoter.

1 1 . A cell as claimed in any of claims 7 to 10, wherein the prokaryotic cell has a hydroxymethyl-selective restriction enzyme system active against hmC and the system is inactivated.

12. A cell which has deoxycytidylate 5-hydroxymethyltransferase (dCMP Hmase) activity and deoxy-Nucleotide Monophosphate (dNMP) kinase activity, and wherein at least a proportion of the cytosine residues of the DNA of the cell are hydroxymethylcytosine (hmC) residues wherein the cell is free of T-even phage infection.

13. A cell which has deoxycytidylate 5-hydroxymethyltransferase (dCMP Hmase) activity, deoxy-Nucleotide Monophosphate (dNMP) kinase activity, and glycosyl transferase activity, wherein at least a proportion of the cytosine residues of the DNA of the cell are glycosyl hydroxymethylcytosine residues.

14. A cell as claimed in claim 12 or claim 13, wherein at least a proportion of the cytosine residues of the DNA of the cell are glucosyl hydroxymethylcytosine (ghmC) residues.

15. A cell as claimed in any preceding claim, wherein the proportion of the

hydroxymethylcytosine residues of the DNA of the cell is at least 90%; preferably at least 98%; optionally 100%.

16. A cell as claimed in any preceding claim, wherein the cell is a prokaryotic cell.

17. A cell as claimed in any preceding claim, which is E. coli.

18. A cell as claimed in claim 17, wherein the E.coli strain is a mutant in which the methylation-requiring restriction system McrBC is inactivated, for example E cloni® 10G, E.coli strain ER1821 , E.coli strain DH10B, E.coli strain T7 Express, ER1793, K803, C2523, or ER2925.

19. A DNA expression construct comprising a promoter and a deoxycytidylate 5- hydroxymethyltransferase (dCMP Hmase) gene and a deoxy-Nucleotide

Monophosphate (dNMP) kinase gene, wherein the genes are under operative control of the promoter.

20. An expression construct as claimed in claim 19, further comprising a

deoxycytidine-triphosphatase (dCTPase) gene under operative control of the promoter.

21 .An expression construct as claimed in claim 19 or claim20, further comprising a glycosyl transferase gene under operative control of the promoter.

22. An expression construct as claimed in claim 21 , wherein the glycosyl transferase gene is a glucosyl transferase gene.

23. A method of preparing DNA, wherein at least a proportion of the cytosine residues are hydroxymethylcytosine (hmC), comprising culturing a cell of any of claims 1 to 1 1 or 12 to 18 for a period, isolating the cell and then extracting DNA from the cell.

24. A method of preparing DNA, wherein at least a proportion of the cytosine

residues of the DNA are glycosyl hydroxymethylcytosine residues, comprising culturing a cell of any of claims 1 to 18 for a period, isolating the cell and then extracting DNA from the cell.

25. A method of preparing DNA as claimed in claim 24, wherein at least a proportion of the cytosine residues of the DNA are glucosyl hydroxymethylcytosine (ghmC) residues.

26. A method as claimed in any of claims 23 to 25, wherein the cell is a prokaryotic cell.

27. A method as claimed in claim 26, wherein the DNA extracted from the cells is plasmid DNA.

28. A method of preparing DNA having at least a proportion of cytosine residues as glycosyl hydroxymethylcytosine residues, comprising contacting isolated DNA having at least a proportion of cytosine residues as hydroxymethylcytosine (hmC) with a glycosyl transferase in the presence of a sugar.

29. A method of preparing DNA as claimed in claim 28, wherein the DNA has at least a proportion of cytosine residues as glucosyl hydroxymethylcytosine (ghmC) residues, comprising contacting isolated DNA having at least a proportion of cytosine residues as hydroxymethylcytosine (hmC) with a glucosyl transferase in the presence of a sugar.

30. A method as claimed in claim 28 or claim 29, wherein the sugar is an activated nucleotide sugar, for example ADP-glucose or UDP-glucose.

31 .A method of transforming a prokaryotic cell with a desired DNA molecule other than by a T-even phage, comprising introducing the desired DNA into the cell, wherein at least a proportion of the cytosine residues of the DNA of the desired DNA molecule are glycosyl hydroxymethylcytosine residues, and wherein the said prokaryotic cell is not transformable with the same desired DNA molecule lacking glycosyl hydroxymethylcytosine residues.

32. A method as claimed in claim 31 , wherein at least a proportion of the cytosine residues of the DNA are glucosyl hydroxymethylcytosine (ghmC) residues.

33. A method as claimed in claim 31 or claim 32, wherein the desired DNA is

comprised in a plasmid produced by a prokaryotic cell of any of claims 6 to 19.

34. A method of genetically modifying a cell by a process involving homologous

recombination, comprising creating a double stranded break at a desired locus and introducing a donor template DNA molecule into the cell, wherein the template DNA has at least a proportion of cytosine residues as

hydroxymethylcytosine (hmC) or glycosyl hydroxymethylcytosine residues.

35. A method as claimed in claim 34, wherein the template DNA has at least one hydroxymethylcytosine (hmC) or glycosyl hydroxymethylcytosine residue in the PAM region and/or protospacer region.

36. A method as claimed in claim 34 or claim 35, wherein the template DNA has at least a proportion of cytosine residues as glucosyl hydroxymethylcytosine (ghmC) residues.

37. A method as claimed in any of claims 34 to 36, wherein the double stranded DNA break is created by a Cas9 nuclease.

38. A method as claimed in any of claims 34 to 37, wherein the cell is a prokaryotic cell.

39. A method as claimed in any of claims 31 to 38, wherein the template DNA is impervious to degradation by sequence-specific nucleases, for example Cas9.