Table 2.
Advantages and disadvantages of different strategies of phage genetic engineering
| Modification strategy | Advantages | Disadvantages |
|---|---|---|
| In vitro modification of phage DNA and transfection of host cells | Phage DNA modification as simple as in the case of plasmids | Applicable mainly to phages of small genomes whose replicative form is circular dsDNA |
| Only GE phage production by transfected cells if the phage is functional | Requires an R-M-deficient and transfection proficient bacterial host | |
| No need for screening or selection | Requires highly competent host cells for transfection with large phage genomes | |
| Host-mediated homologous recombination between bacteriophage DNA and donor DNA cloned in a plasmid | Simplicity | Occurs only with plasmid-cloned DNA fragments as donors for recombination |
| Modification of prophages as efficient as modification of chromosome | Requires an R-M-deficient and transformation proficient bacterial host | |
| Upon induction of prophage lytic development, PCR screening of < 100 plaques typically sufficient to recover recombinants | Recombinant recovery of obligatorily lytic phages so low that requires selection or screening of large number of plaques | |
| As above, but supported by counterselection of unmodified phages with the use of CRISPR-Cas | High efficiency of GE phage recovery | Requires an R-M-deficient and transformation proficient bacterial host |
| Single-step introduction of traceless changes in phage DNA | Requires the presence of active components of CRISPR-Cas system with an appropriate spacer in phage host | |
| No need for selection of GE phages | Modification of various phage genome regions with donor DNA each time requires cloning of relevant spacer in the CRISPR-Cas source plasmid | |
| Suitable for engineering of obligatorily lytic phages, even those that degrade host DNA | ||
| Recombineering with the use of lambda Exo and Beta proteins or their homologs | Occurs with linear dsDNA or ssDNA fragments as donors for recombination | Requires an R-M-deficient and transformation proficient bacterial host |
| Homologous end fragments as short as 50 bp suffice for recombination | Requires the presence of a source of appropriate phage recombination proteins in phage host | |
| Single-step introduction of traceless changes in phage DNA | Low recombinant recovery of obligatorily lytic phages that degrade host DNA | |
| Recombinants of lytic phages that do not degrade host DNA recoverable by PCR screening of plaques | ||
| BRED | As above, but efficiency of modified phage recovery up to 20% | Requires an R-M-deficient bacterial host highly competent for transformation |
| Suitable for engineering obligatorily lytic phages or lytic variants of temperate phages | Requires a source of appropriate phage recombination proteins in phage host | |
| Phage genome rebooting in yeast | Allows introduction of multiple changes to phage DNA or construction fully synthetic phage genomes | Complex methodology and difficulty to assemble large phage genomes |
| Allows independent preparation of various modified phage genome segments for in vivo recombination if they have overlapping ends | Recovery of modified phages requires an R-M-deficient bacterial host highly competent for transformation | |
| Obtaining GE phages impossible if phage genomes recovered from yeast cells are non-functional | ||
| Phage genome rebooting in L-forms of bacteria with Gibson method as an intermediate genome assembly step | Allows modified or fully synthetic phage genomes to be assembled in vitro | Requires R-M-deficient and transformation proficient L-forms of the bacteria supporting intracellular development of phage to be modified |
| Allows the recovery of engineered phages of differing specificities from L-forms of bacteria metabolically compatible with the phage host but not necessarily sensitive to infection with any of the phages | Requires hypotonic lysis of L-forms to release GE phages | |
| Synthesis of engineered phages in a cell-free transcription and translation system (TXTL) | Allows GE phage production in a test tube, independent of phage host | Extremely low efficiency of complete phage synthesis |
| Low cost | Requires high infection efficiency of the host by synthesized phage to allow efficient phage recovery |
BRED bacteriophage recombineering of electroporated DNA, Cas CRISPR-associated proteins, CRISPR clustered regularly interspaced palindromic repeats, dsDNA double-stranded DNA, GE genetically engineered, PCR polymerase chain reaction, R-M restriction-modification, ssDNA single-stranded DNA