CRISPY-BRED and CRISPY-BRIP: efficient bacteriophage engineering

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 bacteriopha...

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Published inScientific reports Vol. 11; no. 1; p. 6796
Main Authors Wetzel, Katherine S, Guerrero-Bustamante, Carlos A, Dedrick, Rebekah M, Ko, Ching-Chung, Freeman, Krista G, Aull, Haley G, Divens, Ashley M, Rock, Jeremy M, Zack, Kira M, Hatfull, Graham F
Format Journal Article
LanguageEnglish
Published England Nature Publishing Group 24.03.2021
Nature Publishing Group UK
Nature Portfolio
Subjects
Bacteriophages - genetics
CRISPR
CRISPR-Cas Systems - genetics
Efficiency
Electroporation
Engineering
Genes
Genetic Engineering - methods
Genome editing
Genomes
Genomics
Mutation
Phages
Proteins
Recombination
RNA, Guide, CRISPR-Cas Systems - metabolism
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Summary: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.
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ISSN:2045-2322
2045-2322
DOI:10.1038/s41598-021-86112-6