Abstract
Genome engineering of bacteriophages provides opportunities for precise genetic dissection and for numerous phage applications including therapy. However, few methods are available for facile construction of unmarked precise deletions, insertions, gene replacements and point mutations in bacteriophages for most bacterial hosts. Here we describe CRISPY-BRED and CRISPY-BRIP, methods for efficient and precise engineering of phages in Mycobacterium species, with applicability to phages of a variety of other hosts. This recombineering approach uses phage-derived recombination proteins and Streptococcus thermophilus CRISPR-Cas9.
Highlights
Genome engineering of bacteriophages provides opportunities for precise genetic dissection and for numerous phage applications including therapy
We previously described Bacteriophage Recombineering of Electroporated DNA (BRED) as a method for engineering Mycobacterium smegmatis phages[5], which has been subsequently adapted for phages of Klebsiella[6], Escherichia coli[7], and Salmonella[8]
The combination of highly efficient recombineering systems and CRISPR-Cas selection has been described for engineering of bacterial g enomes[16], 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[17,18]
Summary
Genome engineering of bacteriophages provides opportunities for precise genetic dissection and for numerous phage applications including therapy. 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 619, and plated for infectious centers on a bacterial lawn. We have combined BRED technology with CRISPR-Cas[9] to facilitate efficient and precise phage genome engineering.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
