Techniques for Editing Phage Genome: Comparison
View Latest Version
Please note this is a comparison between Version 2 by Nora Tang and Version 3 by Nora Tang.

Bacteriophages, abbreviated as “phages”, have been developed as emerging nanoprobes for the detection of a wide variety of biological species, such as biomarker molecules and pathogens. Nanosized phages can display a certain length of exogenous peptides of arbitrary sequence or single-chain variable fragments (scFv) of antibodies that specifically bind to the targets of interest, such as animal cells, bacteria, viruses, and protein molecules. Metal nanoparticles generally have unique plasmon resonance effects. Metal nanoparticles such as gold, silver, and magnetism are widely used in the field of visual detection. A phage can be assembled with metal nanoparticles to form an organic–inorganic hybrid probe due to its nanometer-scale size and excellent modifiability. Due to the unique plasmon resonance effect of this composite probe, this technology can be used to visually detect objects of interest under a dark-field microscope.

  • bacteriophage (phage)
  • biological detection
  • biosensor
  • nanomaterials

1. Homologous Recombination Technique for Editing of Phage Genome

Homologous recombination was the earliest technique used for phage genome modification [1]. Two identical or similar phenotypical parental phage genomes were exchanged based on homologous or similar DNA segments in the host bacterium, and the progeny were screened based on specific phenotypic characteristics. Due to the lack of site specificity, homologous sequences carried by the plasmids are integrated into the phage genome through homologous recombination to obtain the modified phage [2][3]. First, the desired genome was mutated and inserted along with phage homologous DNA into the selected plasmid using standard restriction digestion and ligation, PCR, and site-directed mutagenesis. These genetically modified plasmids were then transformed into host bacteria, which were then injected with phages. The genetically modified phages were screened, but the recombinant phage concentration obtained was very low and the method was time consuming [4][5]. Next, based on efficient recombination engineering and electroporation technology, two main methods were developed for lytic phage recombination: a lambda phage model system and a Mycobacterium phage model system [6][7][8][9][10]. In the lambda phage engineering system, the homologous recombination steps include phage infection, induction of recombination function, competent cell preparation, induction of dsDNA or ssDNA substrate by electroporation, recovery of phage plaques after lysis cycle, and phage mutation analysis. The Mycobacterium recombination engineering system uses a new and potentially powerful method called phage recombineering of electroporated DNA (PRED). Phage recombination has been proven to be an efficient method to construct gene deletions, small fragment insertions, gene replacement, and point mutations in lytic phages, though the recombination rate still needs to be improved. Homologous recombination technology has a wide application prospect; in 2021, Erickson et al. isolated a novel bacteriophage named L. grayi bacteriophage (LPJP1). LPJP1 is the first and only reported jumbo bacteriophage infecting the Listeria genus. This team used homologous recombination technology to reprogram LPJP1, which encodes NanoLuc luciferase, and successfully detected two subtypes of Listeria in just four hours [11]. In the same year, Yoshimitsu Masuda et al. introduced an LLB-producing phage (LLB-phage). Using genetic engineering to introduce the LLB structural gene into the lytic phage genome, this team successfully constructed a novel antimicrobial agent [12].

2. CRISPR-Cas System for Editing of Phage Genome

The recently discovered clustered regularly interspaced short palindromic repeats-associated protein (CRISPR-Cas) system is involved in the natural protection of prokaryotes from foreign DNA invasion [13][14][15]. CRISPR-Cas can be used to edit phage genomes, thereby conferring the advantage of specific production of the expected recombinant phages, with the removal of Cas protein inhibiting the production of wild-type phages. In recent years, the CRISPR-Cas system has been used for genome editing in several organisms, including phages. In general, the CRISPR-Cas system contains two important components: Cas protein and CRISPR RNA (crRNA). CRISPR-Cas for editing the phage genome involves three steps: (a) adaptation, (b) crRNA biogenesis, and (c) interference [16]. In the adaptation step, a short foreign nucleotide sequence (30–40 bp) called the “spacer or tracer” sequence is integrated with CRISPR loci of partially palindromic DNA repeats. In the crRNA biogenesis step, the spacer and partially palindromic DNA repeats are transcribed into crRNA. In the interference step, the crRNA combines with one or more CRISPR associated proteins (Cas) to form a complex called a “protospacer”, which will recognize the DNA and finally degrade it. Based on Cas protein function, CRISPR-Cas systems are classified into six types I to VI, with each of them having several subtypes based on genetic diversity. According to the current scenario, these six types belong to two major classes: the class 1 system encodes multiple Cas proteins involved in types I, III, and IV, and the class 2 system encodes a single Cas protein function to degrade target DNA in type II, V, and VI. In recent years, types I, II, and III CRISPR systems have been mainly used for phage genome editing [17]. The class 1 subtype I-E CRISPR-Cas system from Vibrio cholerae and E. coli and the class 2 subtype II-A system from Streptococcus thermophilus have been used to edit the genome of lytic phages. The CRISPR-Cas system was first used to delete gene 1.7 of T7 phage [18]. In the same year, the class 2 subtype II-A system from S. thermophilus was used for the first time [19]. In 2016, the V. cholerae phage was edited with a class 1 subtype I-E CRISPR-Cas system, which contains both donor DNA and the CRISPR-Cas gene, to successfully obtain recombinant phages [20]. The CRISPR-Cas9 protein system belongs to the type II CRISPR system, which requires a protospacer-adjacent motif (PAM) site to degrade foreign DNA [21][22][23]. The Streptococcus pyogenes Cas9 (SpCas9) protein has a special programmed PAM site containing three base pairs (5′-NRG-3′) to cleave target foreign DNA [24][25]. The sequence 5′-NRG-3, where N = any nucleotide A, T, G, or C, and R = G or A, presents a range of possible SpCas9 PAM sites: AGG, TGG, GGG, CGG, AAG, TAG, GAG, and CAG.
SpCas9 targets and cleaves DNA with the help of the PAM site and spacer sequence already encoded in crRNA. The CRISPR-Cas system with SpCas9 has been used to edit several organisms, including phage genomes. When using this system, Cas9, crRNA, and tracr-RNA (trans-activating crRNA) were simultaneously cloned into the same plasmid [26][27][28]. When the crRNA and tracr-RNA are fused to generate a single guide RNA (sgRNA), the properly prepared highly active sgRNA yields positive results. After the E. coli with CRISPR-Cas9-sgRNA plasmid is infected with M13KO7 phage (Figure 1), the CRISPR-Cas9 plasmids synthesize crRNA and Cas9 protein, forming the effector Cas9-sgRNA complex. This complex has an ability to bind to the M13KO7 phage DNA at the PAM site and create a double-strand break. The broken M13KO7 phage DNA could then be repaired by the donor plasmid with Gene X in the middle of the short (40–60 bp) homologous sequence of gVIII (excluding PAM site) based on the DNA repair pathways of nonhomologous end joining or homologous DNA repair. After this repair pathway, gene insertion results in the production of the recombinant M13KO7 phage with the gene X peptide at the pVIII N-terminal. Unfortunately, a weak sgRNA may lead to false-positive results. The variable activity of crRNA has already been observed in Type I-E and Type IA CRISPR-Cas systems. Since the exact distribution of sgRNA activity in phages is still highly unknown, the prediction of high activity sgRNA in phages currently uses the sgRNA prediction tool based on a eukaryotic dataset [29][30][31]. Conventional recombination technology is highly expensive and time consuming for gene editing, which requires a few rounds of PCR, restriction enzyme digestion, and ligation [32]. These steps have been eliminated by the introduction of oligonucleotide recombination of the template through CRISPR-Cas systems, but the synthesis of long oligonucleotides still has limitations [33]. In 2018, the CRISPR-Cas system from Listeria monocytogenes was used for Listeria phage genome editing [34]. Later, the Type III CRISPR-Cas10 system was also developed and used for editing the staphylococcal phage genome [35]. The CRISPR-Cas10 system provides more protection to the host bacteria by exhibiting high cleavage activity toward the staphylococcal phage.
Biosensors 12 00030 g010 550
Figure 1. Overview of M13KO7 phage genome editing by CRISPR-Cas9. (a) The CRISPR-Cas9 plasmid synthesizes crRNA and CAS9 protein in E. coli, and forms the effector CAS9-crRNA complex; (b) E. coli carrying the CRISPR-Cas9-SgrNA plasmid is infected with phage M13KO7, and the CAS9-CrRNA complex binds to Phage M13KO7 DNA containing PAM sites; (c) Cas9-crRNA complex forms a double-strand break inM13KO7 phage DNA; (d) Bacteria use the donor plasmid carrying the novel gene to repais the broken M13KO7 phage DNA; (e) Generation of the recombinant M13KO7 phage carrying gene X. (f) After translation and packaging, the recombinant M13KO7 phages-expressing gene X is secreted from E. coli.

References

  1. Karam, J.D. Molecular Biology of Bacteriophage T4 Washington; American Society for Microbiology: Washington, DC, USA, 1994.
  2. Oda, M.; Morita, M.; Unno, H.; Tanji, Y. Rapid detection of Escherichia coli O157:H7 by using green fluorescent protein-labeled PP01 bacteriophage. Appl. Environ. Microbiol. 2004, 70, 527–534.
  3. Namura, M.; Hijikata, T.; Miyanaga, K.; Tanji, Y. Detection of Escherichia coli with fluorescent labeled phages that have a broad host range to E. coli in sewage water. Biotechnol. Prog. 2008, 24, 481–486.
  4. Sarkis, G.J.; Jacobs, W.R., Jr.; Hatfull, G.F. L5 luciferase reporter mycobacteriophages: A sensitive tool for the detection and assay of live mycobacteria. Mol. Microbiol. 1995, 15, 1055–1067.
  5. Rao, V.B.; Mitchell, M.S. The N-terminal ATPase site in the large terminase protein gp17 is critically required for DNA packaging in bacteriophage T4. J. Mol. Biol. 2001, 314, 401–411.
  6. Marinelli, L.J.; Piuri, M.; Swigonova, Z.; Balachandran, A.; Oldfield, L.M.; Van Kessel, J.C.; Hatfull, G.F. BRED: A simple and powerful tool for constructing mutant and recombinant bacteriophage genomes. PLoS ONE 2008, 3, e3957.
  7. Thomason, L.C.; Oppenheim, A.B.; Court, D.L. Modifying bacteriophage lambda with recombineering. Methods Mol. Biol. 2009, 501, 239–251.
  8. Marinelli, L.J.; Hatfull, G.F.; Piuri, M. Recombineering: A powerful tool for modification of bacteriophage genomes. Bacteriophage 2012, 2, 5–14.
  9. Murphy, K.C. Phage recombinases and their applications. Adv. Virus Res. 2012, 83, 367–414.
  10. Nafissi, N.; Slavcev, R. Bacteriophage recombination systems and biotechnical applications. Appl. Microbiol. Biotechnol. 2014, 98, 2841–2851.
  11. Erickson, S.; Paulson, J.; Brown, M.; Hahn, W.; Gil, J.; Barron-Montenegro, R.; Moreno-Switt, A.I.; Eisenberg, M.; & Nguyen, M.M. Isolation and engineering of a Listeria grayi bacteriophage. Sci. Rep. 2021, 11, 18947.
  12. Masuda, Y.; Kawabata, S.; Uedoi, T.; Honjoh, K.I.; Miyamoto, T. Construction of Leaderless-Bacteriocin-Producing Bacteriophage Targeting, E. coli and Neighboring Gram-Positive Pathogens. Microbiol. Spectr. 2021, 9, e0014121.
  13. Haft, D.H.; Selengut, J.; Mongodin, E.F.; Nelson, K.E. A Guild of 45 CRISPR-Associated (Cas) Protein Families and Multiple CRISPR/Cas Subtypes Exist in Prokaryotic Genomes. PLoS Comput. Biol. 2005, 1, 60.
  14. Godde, J.S.; Bickerton, A. The Repetitive DNA Elements Called CRISPRs and Their Associated Genes: Evidence of Horizontal Transfer among Prokaryotes. J. Mol. Evol. 2006, 62, 718–729.
  15. Barrangou, R.; Fremaux, C.; Deveau, H.; Richards, M.; Boyaval, P.; Moineau, S.; Romero, D.A.; Horvath, P. CRISPR Provides Acquired Resistance against Viruses in Prokaryotes. Science 2007, 315, 1709–1712.
  16. Hatoum-Aslan, A. Phage Genetic Engineering Using CRISPR⁻Cas Systems. Viruse 2018, 10, 335.
  17. Koonin, E.V.; Makarova, K.S.; Zhang, F. Diversity, classification and evolution of CRISPR-Cas systems. Curr. Opin. Microbiol. 2017, 37, 67–78.
  18. Kiro, R.; Shitrit, D.; Qimron, U. Efficient engineering of a bacteriophage genome using the type I-E CRISPR-Cas system. RNA Biol. 2014, 11, 42–44.
  19. Martel, B.; Moineau, S. CRISPR-Cas: An efficient tool for genome engineering of virulent bacteriophages. Nucleic Acids Res. 2014, 42, 9504–9513.
  20. Box, A.M.; McGuffie, M.J.; O’Hara, B.J.; Seed, K.D. Functional analysis of bacteriophage immunity through a Type I-E CRISPR-Cas system in Vibrio cholerae and its application in bacteriophage genome engineering. J. Bacteriol. 2015, 198, 578–590.
  21. Garneau, J.E.; Dupuis, M.E.; Villion, M.; Romero, D.A.; Barrangou, R.; Boyaval, P.; Fremaux, C.; Horvath, P.; Magadan, A.H.; Moineau, S. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 2010, 468, 67–71.
  22. Deltcheva, E.; Chylinski, K.; Sharma, C.M.; Gonzales, K.; Chao, Y.; Pirzada, Z.A.; Eckert, M.R.; Vogel, J.; Charpentier, E. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 2011, 471, 602–607.
  23. Shah, S.A.; Erdmann, S.; Mojica, F.J.M.; Garrett, R.A. Protospacer recognition motifs: Mixed identities and functional diversity. RNA Biol. 2013, 10, 891–899.
  24. Hsu, P.D.; Scott, D.A.; Weinstein, J.A.; Ran, F.A.; Konermann, S.; Agarwala, V.; Li, Y.; Fine, E.J.; Wu, X.; Shalem, O.; et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 2013, 31, 827–832.
  25. Jiang, W.; Bikard, D.; Cox, D.; Zhang, F.; Marraffini, L.A. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat. Biotechnol. 2013, 31, 233–239.
  26. Mohanraju, P.; Makarova, K.S.; Zetsche, B.; Zhang, F.; Koonin, E.V.; van der Oost, J. Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems. Science 2016, 353, 5147.
  27. Lemay, M.L.; Tremblay, D.M.; Moineau, S. Genome Engineering of Virulent Lactococcal Phages Using CRISPR-Cas9. ACS Synth. Biol. 2017, 21, 1351–1358.
  28. Tao, P.; Wu, X.; Tang, W.C.; Zhu, J.; Rao, V. Engineering of Bacteriophage T4 Genome Using CRISPR-Cas9. ACS Synth. Biol. 2017, 6, 1952–1961.
  29. Lee, C.M.; Davis, T.H.; Bao, G. Examination of CRISPR/Cas9 design tools and the effect of target site accessibility on Cas9 activity. Exp. Physiol. 2017, 103, 456–460.
  30. Mohr, S.E.; Hu, Y.; Ewen-Campen, B.; Housden, B.E.; Viswanatha, R.; Perrimon, N. CRISPR guide RNA design for research applications. FEBS J. 2016, 283, 3232–3238.
  31. Dang, Y.; Jia, G.; Choi, J.; Ma, H.; Anaya, E.; Ye, C.; Shankar, P.; Wu, H. Optimizing sgRNA structure to improve CRISPR-Cas9 knockout efficiency. Genome Biol. 2015, 16, 280.
  32. Sharan, S.K.; Thomason, L.C.; Kuznetsov, S.G.; Court, D.L. Recombineering: A homologous recombination-based method of genetic engineering. Nat. Protoc. 2009, 4, 206–223.
  33. Kosuri, S.; Church, G.M. Large-scale de novo DNA synthesis: Technologies and applications. Nat. Methods 2014, 11, 499–507.
  34. Hupfeld, M.; Trasanidou, D.; Ramazzini, L.; Klumpp, J.; Loessner, M.J.; Kilcher, S. A functional type II-A CRISPR-Cas system from Listeria enables efficient genome editing of large non-integrating bacteriophage. Nucleic Acids Res. 2018, 46, 6920–6933.
  35. Bari, S.M.N.; Walker, F.C.; Cater, K.; Aslan, B.; Hatoum-Aslan, A. Strategies for Editing Virulent Staphylococcal Phages Using CRISPR-Cas10. ACS Synth. Biol. 2017, 6, 2316–2325.
More
© Text is available under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/)
ScholarVision Creations
Feedback