Name: Precise Phage Mutagenesis with NgTET-Assisted CRISPR-Cas Systems (Video) | JoVE Journal
Uploaded: 2025-10-14T13:20:00.000+00:00
Duration: 10 min 52 s
Description: Learn precise CRISPR-Cas mutagenesis in bacteriophages using NgTET enzyme to reduce DNA modifications. This scarless method enables efficient phage engineering for biotechnology and therapeutic applications.

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5 minutes

This project has received funding from the German Research Council (DFG; SPP 2330 project number 464500427, RTG 2355 project 11, and RTG 2937 (project number 505997786) from the Max Planck Society (Max Planck Research Group Leader funding to K.H.).

<ol><li>Pozhydaieva, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Temporal+epigenome+modulation+enables+efficient+bacteriophage+engineering+and+functional+analysis+of+phage+DNA+modifications%5BTitle%5D)+AND+%22PLoS+Genet%22%5BJournal%5D)">Temporal epigenome modulation enables efficient bacteriophage engineering and functional analysis of phage DNA modifications.</a> PLoS Genet. 20 (9), e1011384(2024).</li>
<li>Schroven, K., Aertsen, A., Lavigne, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bacteriophages+as+drivers+of+bacterial+virulence+and+their+potential+for+biotechnological+exploitation%5BTitle%5D)+AND+%22FEMS+Microbiol+Rev%22%5BJournal%5D)">Bacteriophages as drivers of bacterial virulence and their potential for biotechnological exploitation.</a> FEMS Microbiol Rev. 45 (1), fuaa041(2021).</li>
<li>Moye, Z. D., Woolston, J., Sulakvelidze, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bacteriophage+applications+for+food+production+and+processing%5BTitle%5D)+AND+%22Viruses%22%5BJournal%5D)">Bacteriophage applications for food production and processing.</a> Viruses. 10 (4), 205(2018).</li>
<li>Wolfram-Schauerte, M., Pozhydaieva, N., Viering, M., Glatter, T., Hofer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Integrated+omics+reveal+time-resolved+insights+into+T4+phage+infection+of+E.+coli+on+proteome+and+transcriptome+levels%5BTitle%5D)+AND+%22Viruses%22%5BJournal%5D)">Integrated omics reveal time-resolved insights into T4 phage infection of E. coli on proteome and transcriptome levels.</a> Viruses. 14 (11), 2502(2022).</li>
<li>Usman, S. S., Uba, A. I., Christina, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bacteriophage+genome+engineering+for+phage+therapy+to+combat+bacterial+antimicrobial+resistance+as+an+alternative+to+antibiotics%5BTitle%5D)+AND+%22Mol+Biol+Rep%22%5BJournal%5D)">Bacteriophage genome engineering for phage therapy to combat bacterial antimicrobial resistance as an alternative to antibiotics.</a> Mol Biol Rep. 50 (8), 7055-7067 (2023).</li>
<li>Vlot, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bacteriophage+DNA+glucosylation+impairs+target+DNA+binding+by+type+I+and+II+but+not+by+type+V+CRISPR-Cas+effector+complexes%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">Bacteriophage DNA glucosylation impairs target DNA binding by type I and II but not by type V CRISPR-Cas effector complexes.</a> Nucleic Acids Res. 46 (2), 873-885 (2018).</li>
<li>Tao, P., Wu, X., Tang, W. C., Zhu, J., Rao, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engineering+of+bacteriophage+T4+genome+using+CRISPR-Cas9%5BTitle%5D)+AND+%22ACS+Synth+Biol%22%5BJournal%5D)">Engineering of bacteriophage T4 genome using CRISPR-Cas9.</a> ACS Synth Biol. 6 (10), 1952-1961 (2017).</li>
<li>Mohanraju, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diverse+evolutionary+roots+and+mechanistic+variations+of+the+CRISPR-Cas+systems%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems.</a> Science. 353 (6299), aad5147(2016).</li>
<li>Zemach, A., McDaniel, I. E., Silva, P., Zilberman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome-wide+evolutionary+analysis+of+eukaryotic+DNA+methylation%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Genome-wide evolutionary analysis of eukaryotic DNA methylation.</a> Science. 328 (5980), 916-919 (2010).</li>
<li>Hashimoto, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structure+of+a+Naegleria+Tet-like+dioxygenase+in+complex+with+5-methylcytosine+DNA%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Structure of a Naegleria Tet-like dioxygenase in complex with 5-methylcytosine DNA.</a> Nature. 506 (7488), 391-395 (2014).</li>
<li>Shitrit, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+engineering+of+marine+cyanophages+reveals+integration+but+not+lysogeny+in+T7-like+cyanophages%5BTitle%5D)+AND+%22ISME+J%22%5BJournal%5D)">Genetic engineering of marine cyanophages reveals integration but not lysogeny in T7-like cyanophages.</a> ISME J. 16 (2), 488-499 (2022).</li>
<li>Dedrick, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engineered+bacteriophages+for+treatment+of+a+patient+with+a+disseminated+drug-resistant+Mycobacterium+abscessus%5BTitle%5D)+AND+%22Nat+Med%22%5BJournal%5D)">Engineered bacteriophages for treatment of a patient with a disseminated drug-resistant Mycobacterium abscessus.</a> Nat Med. 25 (5), 730-733 (2019).</li>
<li>Marinelli, L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((BRED%3A+a+simple+and+powerful+tool+for+constructing+mutant+and+recombinant+bacteriophage+genomes%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">BRED: a simple and powerful tool for constructing mutant and recombinant bacteriophage genomes.</a> PLoS One. 3 (12), e3957(2008).</li>
<li>Adler, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Broad-spectrum+CRISPR-Cas13a+enables+efficient+phage+genome+editing%5BTitle%5D)+AND+%22Nat+Microbiol%22%5BJournal%5D)">Broad-spectrum CRISPR-Cas13a enables efficient phage genome editing.</a> Nat Microbiol. 7 (12), 1967-1979 (2022).</li>
<li>Pozhydaieva, N., Höfer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Utilizing+Golden+Gate+assembly+to+streamline+CRISPR-Cas%2FNgTET-based+phage+mutagenesis%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Utilizing Golden Gate assembly to streamline CRISPR-Cas/NgTET-based phage mutagenesis.</a> Methods Mol Biol. 2850, 329-343 (2025).</li>
<li>Ramírez Rojas, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DuBA.Flow%E2%94%80a+low-cost%2C+long-read+amplicon+sequencing+workflow+for+the+validation+of+synthetic+DNA+constructs%5BTitle%5D)+AND+%22ACS+Synth+Biol%22%5BJournal%5D)">DuBA.Flow─a low-cost, long-read amplicon sequencing workflow for the validation of synthetic DNA constructs.</a> ACS Synth Biol. 13 (2), 457-465 (2024).</li>
<li>Lagerbäck, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bacteriophage+T4+endonuclease+II%2C+a+promiscuous+GIY-YIG+nuclease%2C+binds+as+a+tetramer+to+two+DNA+substrates%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">Bacteriophage T4 endonuclease II, a promiscuous GIY-YIG nuclease, binds as a tetramer to two DNA substrates.</a> Nucleic Acids Res. 37 (18), 6174-6183 (2009).</li>
<li>Lagerbäck, P., Carlson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Amino+acid+residues+in+the+GIY-YIG+endonuclease+II+of+phage+T4+affecting+sequence+recognition+and+binding+as+well+as+catalysis%5BTitle%5D)+AND+%22J+Bacteriol%22%5BJournal%5D)">Amino acid residues in the GIY-YIG endonuclease II of phage T4 affecting sequence recognition and binding as well as catalysis.</a> J Bacteriol. 190 (16), 5533-5544 (2008).</li>
<li>Shembrey, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Principles+of+CRISPR-Cas13+mismatch+intolerance+enable+selective+silencing+of+point-mutated+oncogenic+RNA+with+single-base+precision%5BTitle%5D)+AND+%22Sci+Adv%22%5BJournal%5D)">Principles of CRISPR-Cas13 mismatch intolerance enable selective silencing of point-mutated oncogenic RNA with single-base precision.</a> Sci Adv. 10 (51), eadl0731(2024).</li>
<li>Molina Vargas, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((New+design+strategies+for+ultra-specific+CRISPR-Cas13a-based+RNA+detection+with+single-nucleotide+mismatch+sensitivity%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">New design strategies for ultra-specific CRISPR-Cas13a-based RNA detection with single-nucleotide mismatch sensitivity.</a> Nucleic Acids Res. 52 (2), 921-939 (2024).</li>
<li>Shi, P., Wu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Programmable+RNA+targeting+with+CRISPR-Cas13%5BTitle%5D)+AND+%22RNA+Biol%22%5BJournal%5D)">Programmable RNA targeting with CRISPR-Cas13.</a> RNA Biol. 21 (1), 1-9 (2024).</li>
<li>Levin, B. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+population+and+evolutionary+dynamics+of+phage+and+bacteria+with+CRISPR-mediated+immunity%5BTitle%5D)+AND+%22PLoS+Genet%22%5BJournal%5D)">The population and evolutionary dynamics of phage and bacteria with CRISPR-mediated immunity.</a> PLoS Genet. 9 (3), e1003312(2013).</li>
<li>Hershey, A. D., Chase, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Independent+functions+of+viral+protein+and+nucleic+acid+in+growth+of+bacteriophage%5BTitle%5D)+AND+%22J+Gen+Physiol%22%5BJournal%5D)">Independent functions of viral protein and nucleic acid in growth of bacteriophage.</a> J Gen Physiol. 36, 39-56 (1952).</li>
<li>Bair, C. L., Rifat, D., Black, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Exclusion+of+glucosyl-hydroxymethylcytosine+DNA+containing+bacteriophages+is+overcome+by+the+injected+protein+inhibitor+IPI%5BTitle%5D)+AND+%22J+Mol+Biol%22%5BJournal%5D)">Exclusion of glucosyl-hydroxymethylcytosine DNA containing bacteriophages is overcome by the injected protein inhibitor IPI.</a> J Mol Biol. 366 (3), 779-789 (2007).</li>
</ol>

KH and NP filed a European Patent Application for "Engineering of Phages", European Patent Application No. 23 175 257.7. The other authors declare no competing interests.

This study presents a robust and adaptable strategy for introducing precise point mutations into bacteriophage genomes, including single-nucleotide changes. A central element of the workflow is the integration of stringent validation steps at each stage, which is essential for ensuring both the accuracy and efficiency of genome editing. This is particularly critical given the multistep nature of the workflow and the dynamic interactions between phages and their bacterial hosts, which introduce numerous variables that can...

Bacteriophages are viruses that specifically target bacteria. They have attracted significant research interest due to their immense potential in biotechnology and medicine, particularly in the fight against multidrug-resistant bacteria1,2,3. Despite this, phage biology remains relatively understudied. A deeper understanding of their molecular mechanisms of hijacking their bacterial hosts is essential to fully harness their therapeutic and biotechnological potential4. To investigate the molecular mechanisms underlying the efficient phage infection and further to engineer the phages for specific applications, the ability to genetically modify phage genomes is essential. Yet it remains one of the most significant challenges in the field of phage biology3,5,6.
Phage DNA is often modified by specific chemical alterations of the purine and pyrimidine bases, e.g., cytosine glycosylation and methylation. These modifications protect phage genetic material from recognition and degradation by host nucleases6. While these modifications are crucial for the phage fitness, they are a significant obstacle for phage genome engineering approaches that rely on DNA targeting systems2,3,7.
Existing mutagenesis strategies, including restriction-modification systems and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) protein-based technologies, face significant challenges when modifying phage DNA due to protective epigenetic modifications like cytosine glycosylation8. CRISPR-Cas-mediated genome editing, which is highly effective in bacterial and eukaryotic cells, relies on two key steps: the precise cleavage of DNA at targeted genomic loci by the Cas nuclease, followed by DNA repair through homologous recombination with donor DNA carrying the desired mutation1. However, in phages, DNA modifications often prevent Cas nucleases from binding or cleaving the genome efficiently. Additionally, the rapid and transient nature of phage replication can further limit the efficiency of homologous recombination. These barriers make it particularly difficult to apply DNA-targeting CRISPR-Cas technology for phage mutagenesis. Phage DNA modifications have been shown to impair phage DNA targeting with CRISPR-Cas both in vitro and in vivo6,7.
To address the challenges posed by protective DNA modifications in phages, we introduce a genome mutagenesis strategy that harnesses the activity of the ten-eleven translocation (TET) methylcytosine dioxygenase, specifically NgTET. In eukaryotes, TET enzymes catalyze the stepwise oxidation of methylated cytosine through iterative processes: methylcytosine (5mdC) is first converted to hydroxymethylcytosine (5hmdC), then to formylcytosine (5fdC), and finally to carboxycytosine (5cadC) (Figure 1).
In our phage mutagenesis approach, NgTET is used to temporarily reduce the abundance of protective DNA modifications, thereby enhancing the accessibility of phage DNA to genome editing tools such as Cas enzymes9. NgTET, was selected for modulating the bacteriophage genome due to previous reports of its successful expression in active soluble form in a heterologous bacterial host, particularly E. coli. This property is essential, as NgTET must remain active during phage infection10.
<img alt="Eukaryotic dC metabolism by TET dioxygenases; decarboxylation process; chemical structure diagram." class="xfigimg" src="/files/ftp_upload/69022/69022fig1v2.jpg" data-altupdatedat="1783396841000" data-alt="Figure 1"> 
Figure 1: Stepwise Oxidation of 5-Methylcytosine by TET Dioxygenase1. The TET dioxygenase catalyzes the successive oxidation of 5-methyl-2´-deoxycytidine (5mdC) to 5hmdC, 5-formyl-2´-deoxycytidine (5fdC), and ultimately to 5-carboxyl-2´-deoxycytidine (5cadC). The final product, 5cadC, either spontaneously or enzymatically reverts to unmodified cytosine (dC), thereby contributing to dynamic epigenetic regulation. This multistep process is central to active DNA methylation in eukaryotes. <a href="https://www.jove.com/files/ftp_upload/69022/69022fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Since methylcytosine and hydroxymethylcytosine are common precursors to phage DNA hypermodifications, including bulky protective structures like glycosylations, the oxidative activity of TET dioxygenase can be exploited to prevent the formation of these DNA hypermodifications9. In this TET-based genome mutagenesis approach, we heterologously express NgTET recombinantly in E. coli to oxidize hydroxymethylated cytosines in the phage genome upon infection, thereby preventing the formation of bulky glycosylation. By decreasing the abundance of these bulky DNA modifications, our approach can increase phage DNA accessibility to CRISPR-Cas nucleases. This enhanced accessibility facilitates efficient target recognition, precise DNA cleavage, and the introduction of scarless point mutations into the phage genome, distinguishing it from traditional phage mutagenesis methods that rely on gene deletions, reporter gene insertions, or the integration of artificial junctions for PCR-based mutant selection1,11. These conventional strategies have so far been the only available tools for targeted mutagenesis in phage genomes and therefore represent an important foundation for the method presented here. However, single phage proteins can possess multiple functions. Deletion of the entire gene encoding a protein of interest disrupts all associated functions. In contrast, the targeted introduction of point mutations permits the selective inactivation of specific functions, enabling detailed analysis of their roles. To date, the functions of many phage proteins remain largely uncharacterized; thus, we may overlook additional activities linked to a given gene. Therefore, a minimalistic approach that aims to remove only a single function while causing minimal changes to the overall protein is preferred12,13. The method described here enables precise genetic modifications without disrupting the overall genomic organization or function. Another major challenge of previous phage mutagenesis methods has been their low efficiency, which typically yielded mutation rates of around 0.1%1,11. With this technique, we present the first mutagenesis strategy capable of introducing single-codon changes, achieving a notable sevenfold increase in targeting efficiency. By combining it with an ONT-based high-throughput screening method, the detection of point mutations is simplified, eliminating the need to screen large phage populations to isolate one mutant. The TET-based mutagenesis strategy described here addresses a longstanding limitation in the field and enables more reliable genome editing14.
Overall, this method cannot only be applied to advance our understanding of bacteriophage infection mechanisms but also holds significant promise for synthetic biology. By enabling precise and efficient phage genome engineering, it paves the way for tailoring the phages to specific applications, thereby enhancing their potential for biotechnological and medical applications.

<table><tbody><tr><td>1.5 mL reaction tubes</td><td>Sarstedt</td><td>72.690.001</td><td></td></tr><tr><td>15 mL Canocial Tubes</td><td>Sarstedt</td><td>62.554.502</td><td></td></tr><tr><td>15 mL Open-Top Thinwall Polypropylene Tubes</td><td>Beckman Coulter Life Sciences</td><td>361706</td><td>16 mm x 95 mm (for ultracentrifugation)</td></tr><tr><td>50 mL canonical tubes</td><td>Sarstedt</td><td>62.547.254</td><td></td></tr><tr><td>5x GC buffer</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>F519L</td><td></td></tr><tr><td>6x TriTrack DNA Loading Dye&amp;nbsp;</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>R1161</td><td></td></tr><tr><td>Agarose</td><td>Meridian BIOSCIENCE</td><td>BIO-41025</td><td>Molecular Grade</td></tr><tr><td>Avanti J-30I Centrifuge</td><td>Beckman Coulter Life Sciences</td><td>no catalog&amp;nbsp; number</td><td>Serien-Nr. JKY14M01, https://www.beckman.de/centrifuges/ultracentrifuges/optima-xe</td></tr><tr><td>Boric acid&amp;nbsp;</td><td>Merck</td><td>10043-35-3</td><td></td></tr><tr><td>BsaI-HF v2</td><td>NEB</td><td>R3733S</td><td></td></tr><tr><td>Calciumchlorid Hexahydrat</td><td>ACROS ORGANICS</td><td>7774-34-7</td><td>97 %, extra pure&amp;nbsp;</td></tr><tr><td>Cell density meter</td><td>Harvard Biochrom&amp;nbsp;</td><td>80211630</td><td></td></tr><tr><td>ChemiDoc MP&amp;nbsp;</td><td>Biorad</td><td>12003154</td><td></td></tr><tr><td>Chloroform</td><td>VWR</td><td>650498</td><td></td></tr><tr><td>Cuvettes</td><td>Sarstedt</td><td>67.742</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)&amp;nbsp;</td><td>Sigma Aldrich</td><td>D4540</td><td></td></tr><tr><td>Di-Sodium hydrogen phosphate</td><td>neoFroxx GmbH</td><td>7558-79-4</td><td>pharma grade</td></tr><tr><td>Disposable Glass Pasteur Pipettes</td><td>VWR</td><td>612-1702</td><td>230 mm</td></tr><tr><td>DNase I&amp;nbsp;</td><td>Roche</td><td>4716728001</td><td>10 U/&amp;micro;L</td></tr><tr><td>dNTP mix (10 mM each dNTP)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>72004</td><td></td></tr><tr><td>DS_SPcas_ModA</td><td>In-house product.</td><td>https://github.com/MaikTungsten/CRISPRT4/tree/main/Plasmid_maps</td><td>Plasmid construct that contains CRISPR-Cas12/ModA sgRNA</td></tr><tr><td>DS-spCas (CRISPR-Cas9 system)</td><td>Addgene&amp;nbsp;</td><td>48645 (addgene accessory number)</td><td>Plasmid for Construction of Spacer-Containing CRISPR-Cas Plasmids (CRISPR-Cas9 system) for phage mutagenesis (step 3.2)</td></tr><tr><td>E. coli B Strain Strain was used for T4 infection</td><td>DSMZ</td><td>no catalog&amp;nbsp; number</td><td>Escherichia coli (Migula 1895) Castellani and Chalmers 1919 (DSM 613, ATCC 11303)</td></tr><tr><td>E. coli BL21 (DE3) Expression strain, competent cell</td><td>Invitrogen</td><td>C600003</td><td>Expression strain, competent cells.</td></tr><tr><td>E. coli DH5&amp;alpha; Cloning strain, competent cells</td><td>Invitrogen</td><td>18265017</td><td>Cloning strain, competent cells.&amp;nbsp;</td></tr><tr><td>EDTA</td><td>Roth</td><td>8043.2</td><td></td></tr><tr><td>Ethanol (absolute)&amp;nbsp;</td><td>VWR</td><td>200-578-6</td><td></td></tr><tr><td>Flongle Flow Cell</td><td>Oxford Nanopore Technologies</td><td>no catalog&amp;nbsp; number</td><td>R10.4.1. chemistry, https://nanoporetech.com/document/flongle</td></tr><tr><td>Gene JET Plasmid Miniprep Kit&amp;nbsp;</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>10242490</td><td></td></tr><tr><td>GeneRuler 1 kb Plus DNA ladder</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>SM1331</td><td></td></tr><tr><td>GeneRuler ULR DNA ladder&amp;nbsp;</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>SM1213</td><td></td></tr><tr><td>Gradient Master 108</td><td>BIOCOMP</td><td>no catalog&amp;nbsp; number</td><td>https://biocompinstruments.com/our-approach/gradient-forming</td></tr><tr><td>Heraeus Pico 17 Centrifuge</td><td>Thermo Scientific</td><td>75002410</td><td></td></tr><tr><td>Infors HT Incubator Minitron</td><td>Infors AG</td><td>no catalog&amp;nbsp; number</td><td>https://infors-ht.com/de/produkte/inkubationsschuettler/minitron#productspecs</td></tr><tr><td>Isopropyl-&amp;beta;-D-1-thiogalactopyranoside (IPTG)</td><td>Sigma-Aldrich</td><td>367-93-1</td><td></td></tr><tr><td>Kanamycin</td><td>Carl Roth</td><td>T832.1</td><td></td></tr><tr><td>LB-Agar</td><td>Carl-Roth</td><td>X969.1</td><td></td></tr><tr><td>LB-medium (Luria/Miller)&amp;nbsp;</td><td>Carl-Roth</td><td>X968.1</td><td></td></tr><tr><td>Magnesium chloride hexahydrate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>7791-18-6</td><td></td></tr><tr><td>Magnesium sulfate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>7487-88-9</td><td></td></tr><tr><td>MinION device&amp;nbsp;</td><td>Oxfrod Nanopore Technologies</td><td>no catalog&amp;nbsp; number</td><td>https://store.nanoporetech.com/eu/minion.html</td></tr><tr><td>ModA E165A_fw</td><td>IDT</td><td>GAATTAGTTTCAGATGA&lt;br/&gt; ACAAGCGGTAATGATAC&lt;br/&gt; CAGCT</td><td>Forward primer for introduction of point mutation for ModA E165A and to introduce sticky ends for Golden Gate Cloning (Step 3.1)</td></tr><tr><td>ModA E165A_rev</td><td>IDT</td><td>CATATTACGATAACGA&lt;br/&gt; TGACTATCCGGAAAC</td><td>Reverse primer for introduction of point mutation for ModA E165A and to introduce sticky ends for Golden Gate Cloning (Step 3.1)</td></tr><tr><td>ModA_sgRNA_Cas12_fw</td><td>IDT</td><td>TCTTGTTCATCTACAAC&lt;br/&gt; AGTAGAAATTAATTTAAA&lt;br/&gt; GTTCTTAGACCCG</td><td>Forward primer for generating spacer for CRISPR-Cas12/ CRISPR-Cas9/ CRISPR-Cas13 systems for phage mutagenesis and counterselection (step 3.2 and step 4.1)</td></tr><tr><td>ModA_sgRNA_Cas12_rev</td><td>IDT</td><td>AGTAATGATAAAGAACT&lt;br/&gt; TTAAATAATTTCTACTGT&lt;br/&gt; TGTAGATGCTAC</td><td>Reverse primer for generating spacer for CRISPR-Cas12/ CRISPR-Cas9/ CRISPR-Cas13 systems for phage mutagenesis and counterselection (step 3.2 and step 4.1)</td></tr><tr><td>NanoDrop ND-1000 Spectrophotometer&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>AZY2019944</td><td></td></tr><tr><td>NucleoMag Kit&amp;nbsp;</td><td>Machery Nagel</td><td>REF 744970.50</td><td></td></tr><tr><td>Nucleoside digestion mix&amp;nbsp;</td><td>New England Biolabs</td><td>M0649S</td><td></td></tr><tr><td>pBA560 (CRISPR-Cas13 system)</td><td>Addgene</td><td>186236 (addgene accessory number)</td><td>Plasmid encoding CRISPR-Cas13 system for counterselection (step 4.1)</td></tr><tr><td>pCpfl-sp plasmid (CRISPR-Cas12 system)</td><td>Addgene</td><td>122186</td><td></td></tr><tr><td>pCpfl-sp plasmid (CRISPR-Cas12 system)</td><td>Addgene</td><td>122186 (addgene accessory number)</td><td>Plasmid for Construction of Spacer-Containing CRISPR-Cas Plasmids (CRISPR-Cas12 system) for phage mutagenesis (step 3.2)</td></tr><tr><td>PCR reaction vessels</td><td>Sarstedt</td><td>72.991.002</td><td></td></tr><tr><td>PeqGREEN</td><td>VWR</td><td>732-3196</td><td></td></tr><tr><td>pET28a_NgTET&amp;nbsp;</td><td>In-house product.</td><td>no catalog&amp;nbsp; number</td><td>plasmid is available upon request, the plasmid map can be found at the following https://zenodo.org/records/10615143&amp;nbsp;</td></tr><tr><td>pET28a_NgTET&amp;nbsp;</td><td>In-house product.</td><td>no catalog&amp;nbsp; number</td><td>Plasmid for construction of pET28a_NgTET_donor DNA via Golden Gate Cloning (step 3.1), plasmid&amp;nbsp; is available upon request, the plasmid map can be found at the following https://zenodo.org/records/10615143&amp;nbsp;</td></tr><tr><td>Petri Plates</td><td>Sarstedt</td><td>82.1473.001</td><td>92 x 16 mm with cams</td></tr><tr><td>Phusion DNA Polymerase&amp;nbsp;</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>F-530XL</td><td>2 U/&amp;micro;L</td></tr><tr><td>Potassium Phosphate</td><td>Sigma Aldrich</td><td>7778-77-0</td><td>Monobasic</td></tr><tr><td>PowerPac Universal Power Supply</td><td>Bio Rad</td><td>1645070</td><td></td></tr><tr><td>Proteinase K&amp;nbsp;</td><td>Carl Roth&amp;nbsp;</td><td>7528.1</td><td>100 mg/mL</td></tr><tr><td>QIAquick PCR Purification Kit&amp;nbsp;</td><td>QIAGEN</td><td>28506</td><td></td></tr><tr><td>Quantus Fluormeter, QuantiFluor dsDNA system</td><td>Promega</td><td>E2670</td><td></td></tr><tr><td>RNase A/T1&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>EN0551</td><td>mix 2 mg/mL of RNase A 5000 U/mL of RNase T1&amp;nbsp;</td></tr><tr><td>Roti-Aqua P/C/I for DNA extraction&amp;nbsp;</td><td>Carl Roth</td><td>X985.1</td><td></td></tr><tr><td>Sodium acetate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>127-09-3</td><td></td></tr><tr><td>Sodium chloride&amp;nbsp;</td><td>Sigma-Aldrich</td><td>7647-14-5</td><td></td></tr><tr><td>SQK-LSK109 Ligation sequencing Kit</td><td>Oxford Nanopore Technologies</td><td>no catalog&amp;nbsp; number</td><td>https://nanoporetech.com/document/gDNA-q-sqk-lsk109</td></tr><tr><td>Steritop filters, 0.22 &amp;micro;m pore size</td><td>Millipore</td><td>S2GPT05RE</td><td>250 mL and 500 mL</td></tr><tr><td>Streptomycin 50 mg/L</td><td>Carl Roth</td><td>HP66.1</td><td></td></tr><tr><td>Succrose</td><td>NeoFroxx GmbH</td><td>57-50-1</td><td>for molecular biology&amp;nbsp;</td></tr><tr><td>Swinging-Bucket Aluminum Rotor&amp;nbsp;</td><td>Beckman Coulter Life Sciences</td><td>JS[1]24.15</td><td></td></tr><tr><td>T4 DNA Ligase 5 U/&amp;micro;L</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>EL0011</td><td></td></tr><tr><td>T4 DNA Ligase Buffer</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>B69</td><td></td></tr><tr><td>Test Tubes, heavy wall, rimless</td><td>Karl Hecht GmbH</td><td>42775054</td><td>100 mm x 18 mm</td></tr><tr><td>Thermal cycler, T100</td><td>Biorad</td><td>1861096</td><td></td></tr><tr><td>Thermomixer comfort&amp;nbsp;</td><td>Eppendorf</td><td>5382000015</td><td></td></tr><tr><td>Trizma Base</td><td>Sigma Aldrich</td><td>77-86-1</td><td></td></tr><tr><td>Wather bath, VWB2&amp;nbsp;</td><td>VWR</td><td>462-0557</td><td>12 L volume</td></tr></tbody></table>

precise phage mutagenesis with ngtet-assisted crispr-cas systems

Precise Phage Mutagenesis with NgTET-Assisted CRISPR-Cas Systems

Bacteriophages, viruses that specifically target their bacterial hosts, hold significant potential for biotechnology and medicine, especially in combating multidrug-resistant infections. However, the molecular mechanisms underlying phage infection remain largely underexplored. Precise, site-specific mutagenesis of phages is a powerful tool to elucidate gene functions and phage-host interactions.
However, a major challenge in phage genome mutagenesis is the presence of phage DNA modifications that interfere with conventional genome editing tools like CRISPR-Cas. 
While CRISPR-Cas systems have been used successfully for targeted mutagenesis in various organisms, their effectiveness in phage mutagenesis is often limited by DNA modifications such as cytosine glycosylation. To overcome this barrier, we developed an efficient method that temporarily reduces the abundance of phage DNA modifications, enabling efficient CRISPR-Cas targeting and precise mutation introduction into phage genomes. Specifically, we use the Ten Eleven Translocation (TET) methylcytosine dioxygenase from Naegleria gruberi (NgTET), which iteratively demodifies methylated and hydroxymethylated cytosines in DNA. By oxidizing hydroxymethylated cytosines within phage DNA, NgTET prevents subsequent cytosine modification like glycosylation and significantly enhances the efficiency of Cas-mediated DNA cleavage.
In conclusion, the scarless and precise genome-editing approach presented here enables the efficient introduction of point mutations while maintaining the native gene architecture in phage genomes. By preserving intact transcriptional and translational frameworks, this method minimizes unintended disruptions to complex regulatory networks. This is particularly important for investigating essential or multifunctional phage proteins. The ability to generate targeted genetic modifications without introducing extraneous sequences significantly expands the experimental toolkit for phage biology. This strategy not only facilitates detailed functional studies but also enhances the potential for rational engineering of phages for therapeutic and biotechnological applications.

Identification of the phage band in the gradient. 
The visibility of the phage band in the gradient depends on the phage concentration in the initial sample loaded on the gradient. When the phage is sufficiently concentrated (~&gt;1 × 1011 PFU/mL), the band can be clearly seen by illuminating the gradient from the bottom with a light source (see Figure 3).
However, at low phage concentrations, the band may not be easily visible. In t...

Here, we present a protocol to reduce DNA modifications in bacteriophages using the NgTET enzyme, enabling efficient and scarless CRISPR-Cas mutagenesis. This method facilitates the genetic engineering of phages for applications in biotechnology and phage therapy.

Bacteriophages, viruses that specifically target their bacterial hosts, hold significant potential for biotechnology and medicine, especially in combating ...

We study phage infection at the molecular level. This is really fundamental science. However, we are not only doing the fundamental science, we are also keen to translate this fundamental science into applications where we use this knowledge to study the phage infection, and to use this for phage engineering, and for therapeutic applications.One of the most recent developments in the field of phage research is the discovery of a bacterial immune system. This immune system is present in all bacteria and they identified or generate tools to really fight against the phage. However, phage is quite clever.A phage was able to identify tools as well and to develop tools that allow him to escape those immune systems. And one tool is actually the modification of its DNA by modification. And this is especially for us, really interesting because to study the chemical nature of these DNA modifications that phages have generated to escape immune systems are really interesting, and also allow us to become a tool to engineer the phage at the end.In the last 10 years, wonderful technologies were developed by great colleagues around the whole globe. One of these technologies is for instance, next generation sequencing. Based on this technology, we can really sequence phage, but also the whole host genomes on the molecular level.On the other hand, mass spectrometry, something like proteomics was developed. CRISPR is a wonderful tool that is now applied in various kingdoms of life. And also we are using this now to engineer the phage genome.These are just a few wonderful technologies that have reshaped how we think about phages today. When we talk about current challenges, one of the challenge that we see is the engineering bio CRISPR of a phage genome. The phage genome is totally modified with DNA modifications, and the CRISPR nuclear ACEs are actually not able to cleave the DNA.So this is really a challenge that we need to overcome if we want to engineer the genomes of bacterial phages. My group has discovered a totally novel building block of RNAs. We discovered that specific transcripts are carrying at their five prime terminus, the so-called redox specter, nicotinamide adenine dinucleotide short NAD, and we discovered this also recently now for the first time in E-coli T4 phage interactions.Based on this discovery, we also identified that RNAs and proteins can interact with each other in a totally novel way. We discovered that this NAD that I described before is working like a molecule glue to link RNAs and proteins with each other. This RNA elation is a totally novel way of how RNAs and proteins interact with each other in nature.And we made this discovery in eco anti phage interaction. We are also now keen to translate this fundamental science into applications, for instance, into the field of RNA-based therapeutics. To begin, treat 500 microliters of phage suspension with 20 units of DNA 1.Add two microliters of RNAs AT1 mixed to the tube. Incubate the treated mixture at 37 degrees Celsius for 30 minutes. Next, load 500 microliters of the treated phage solution on top of a prepared sucrose gradient.Centrifuge the gradient at 70, 000 G for 20 minutes at four degrees Celsius. Light the centrifugation tube from the bottom to visualize the turbid phage band in the gradient. Then with a blunt cannula, carefully remove the phage containing fraction into a new ultra ultracentrifugation tube.Now, add 30 milliliters of ice cold TM buffer to the tube and centrifuge. Then resuspend the pellet in 500 microliters of TM buffer after discarding the supernatant. Store the resuspended phages in a glass vial at four degrees Celsius overnight.Add one Microgram of proteinase K to the resuspended phages. Incubate the mixture at 37 degrees Celsius for 30 minutes. Then add an equal volume of phenol chloroform, isoamyl alcohol mixture to the sample.Invert to mix well and centrifuge. Transfer the aqueous phase to a new reaction tube, and repeat the extraction three times. Perform three chloroform back extractions using an equal volume of chloroform to remove residual phenol.Then precipitate the DNA by adding 0.1 volume of three molar sodium acetate at PH 5.5, and 2.5 volumes of ethanol. Incubate the mixture overnight at minus 20 degrees Celsius. The next day, centrifuge the sample at 15, 000 G for one hour at four degrees Celsius to pellet the DNA.Wash the DNA pellet twice with 200 microliters of 70%ethanol. Gently shake the tube and centrifuge again. Carefully remove the supernatant.Then resus, suspend the purified DNA in ultrapure water. Store the DNA at minus 20 degrees Celsius until further use. Inoculate Escherichia coli BL 21D 3 cells that are transformed with both the PET 28 NGTet plasmid, and a CAS 12 or CAS 9 expression plasmid.Grow the culture in LB medium with supplemented with appropriate antibiotics at 37 degrees Celsius with shaking. Induce NG TED expression by adding 0.05 millimolar IPTG, and incubate for an additional two hours at 37 degrees Celsius. As a control, compare E.coli BL 21 D3 strains with and without NgTET overexpression.Transfer 300 microliters of the E.coli cultures into a sterile tube. Infect the culture with either T4 wild type or NgTET treated phage at a multiplicity of infection of 0.01. Mix gently to ensure even phage distribution then incubate again.Add the bacteria phage mixture to four milliliters of LB soft auger containing 0.75%auger and antibiotics. Mix thoroughly, but gently to avoid bubble formation. Pour the soft auger mixture onto a prewarm LB auger plate.Let the plate solidify briefly at room temperature and incubate overnight at 37 degrees Celsius. The next day, count the plaques to determine the number of plaque forming units. For counter selection, infect E.coli cast 13 A spacer using the phages to be counter selected.Perform the counter selection under the same conditions used for mutagenesis. After incubation, filter the SUP natant through a 0.45 micrometer filter to remove bacterial debris. Use the counter selected and filtered phages for a plaque assay on an E.coli B strain to isolate individual plaques for downstream validation.The phage band was clearly visible when the T4 phage sample was sufficiently concentrated. Expression of NgTET protein in E.coli was confirmed by SDS phage showing its presence in both soluble, and insoluble fractions following cell lysis and centrifugation. Chromatograms showed that the relative abundances of dioxiadenosine, diiodohydroxyquinoline, and thymidine were unchanged between wild type and NgTET treated T4 phage DNA.The abundance of modified cytosine derivatives was significantly reduced in the NgTET treated phage DNA compared to the untreated sample. A plasmid containing the NgTET gene, and homology regions for targeted mutagenesis was constructed using golden gate cloning. PCR screening confirmed successful amplification of the target gene mare in lanes two through nine of the agros gel, indicating positive hits among the picked plaques.In wild type T4 phage DNA, the relative abundance of 5 glycosylhydroxyethyl 2 prime deoxidine was high. In NgTET treated T4 phage DNA, levels of 5 glycosylhydroxyethyl 2 prime deoxidine were strongly reduced compared to wild type. T4 phage DNA treated with the catalytic inactive mutant NgTET D234 A retained high levels of 5 glycolhydroxyethyl 2 prime deoxidine.Recovered T 4 phage progeny showed nearly restored levels of 5 hydroxymethyl 2 prime deoxidine compared to wild type.

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Research

JoVE Journal

Bioengineering

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Learn precise CRISPR-Cas mutagenesis in bacteriophages using NgTET enzyme to reduce DNA modifications. This scarless method enables efficient phage engineering for biotechnology and therapeutic applications.

Precise Phage Mutagenesis with NgTET-Assisted CRISPR-Cas Systems

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