Bacteriophage genomics reveals massive genetic diversity and a vast abundance of functionally ill-defined genes. Efficient and precise phage genome engineering is a critical step in understanding phage biology and in developing phages as effective therapeutics, diagnostics, and for numerous other applications. We previously described Bacteriophage Recombineering of Electroporated DNA (BRED) as a method for engineering Mycobacterium smegmatis phages, which has been subsequently adapted for phages of Klebsiella Escherichia coli, and Salmonella. In BRED, phage genomic DNA and a synthetic DNA substrate containing the desired mutation are co-electroporated into bacterial cells that express phage Che9c RecET-like recombination genes,60 and 61, and plated for infectious centers on a bacterial lawn. Recombination is sufficiently efficient to enable identification of plaques containing mutant phage genomes by PCR without genetic selection, although these primary plaques typically contain both mutant and wild type phage particles and require further purification and screening. Deletion of non-essential genes is often simple and relatively efficient using BRED; we previously showed that mixed primary plaques could be recovered from such deletions at an average frequency of 14% (range 4–60%). However, other types of recombinants such as larger deletions, replacements, and insertions are recovered at somewhat lower frequencies, demanding extensive screening of dozens or even hundreds of plaques. This is also observed when genome editing has deleterious impacts on phage growth.
Fig: CRISPY-BRED phage engineering.
CRISPR-Cas systems provide defense against viral attack and are present in numerous bacterial and archaeal species. Spacer sequences between repeat motifs in long arrays are transcribed to produce short RNAs (crRNAs) that together with Cas proteins target invading phage DNA for destruction through recognition of a protospacer corresponding to the crRNA. CRISPR-Cas systems are thus readily adapted for phage engineering, and have been used to modify phages that infect Escherichia coli, Streptococcus thermophilus and Vibrio cholerae among others (Reviewed in Hatoum-Aslan, 2018). These previously described methods primarily rely on host-derived recombination functions and/or CRISPR-Cas. The combination of highly efficient recombineering systems and CRISPR-Cas selection has been described for engineering of bacterial genomes, and we describe a similar approach here for engineering phage genomes, taking advantage of the active and inactive Cas proteins described for genome editing and gene silencing (CRISPRi) in Mycobacterium.
Wetzel, K.S., Guerrero-Bustamante, C.A., Dedrick, R.M. et al. CRISPY-BRED and CRISPY-BRIP: efficient bacteriophage engineering. Sci Rep11, 6796 (2021). https://doi.org/10.1038/s41598-021-86112-6