CRISPY-BRED and CRISPY-BRIP: efficient bacteriophage engineering
Katherine S. Wetzel, Carlos A. Guerrero-Bustamante, Rebekah M. Dedrick, Ching‐Chung Ko, Krista G. Freeman, Haley G. Aull, Ashley M. Divens, Jeremy M. Rock, Kira M. Zack, Graham F. Hatfull
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.