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The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids, forming the foundation for its adaptation as a powerful genome-editing tool. Originally discovered in prokaryotes, this system has been repurposed to revolutionize genetic engineering across a wide range of organisms, including plants, animals, and humans. The core component, Cas9, is an endonuclease derived from Streptococcus pyogenes, capable of introducing...
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Bacteriophages, or phages, are viruses that specifically infect bacteria, utilizing their genetic material to hijack host cellular machinery for replication. DNA bacteriophages employ single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA) genomes. These phages exhibit diverse replication strategies and host interactions, influencing their ecological roles and applications in biotechnology and medicine.ssDNA BacteriophagesssDNA phages, with their small genomes, utilize unique strategies to...
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CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats is a adaptive immune system found in bacteria and archaea that protects against viral infections. This system enables prokaryotic cells to identify, remember, and neutralize foreign genetic elements, primarily bacteriophages, by storing fragments of the invader’s DNA as a genetic memory.The CRISPR immune response begins during an initial infection. Cas (CRISPR-associated) proteins play a central role in this...
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Precise Phage Mutagenesis with NgTET-Assisted CRISPR-Cas Systems
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CRISPY-BRED and CRISPY-BRIP: efficient bacteriophage engineering.

Katherine S Wetzel1, Carlos A Guerrero-Bustamante1, Rebekah M Dedrick1

  • 1Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA, 15260, USA.

Scientific Reports
|March 25, 2021
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Summary
This summary is machine-generated.

New genome engineering methods, CRISPY-BRED and CRISPY-BRIP, enable precise genetic modifications in bacteriophages. These tools facilitate phage therapy and genetic studies across various bacterial hosts.

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Genetics

Background:

  • Bacteriophage genome engineering is crucial for genetic studies and therapeutic applications.
  • Existing methods for precise genetic modifications (deletions, insertions, replacements, point mutations) in bacteriophages are limited, especially for diverse bacterial hosts.

Purpose of the Study:

  • To develop facile and efficient methods for precise, unmarked genome engineering of bacteriophages.
  • To demonstrate the applicability of these methods in Mycobacterium species and potentially other hosts.

Main Methods:

  • CRISPY-BRED and CRISPY-BRIP are novel recombineering-based methods.
  • These methods utilize phage-derived recombination proteins and Streptococcus thermophilus CRISPR-Cas9 system.

Main Results:

  • Successful implementation of CRISPY-BRED and CRISPY-BRIP for precise genome engineering in bacteriophages.
  • Demonstrated efficiency and precision in constructing unmarked deletions, insertions, gene replacements, and point mutations.
  • Applicability shown in Mycobacterium species, suggesting broader utility for other phage-host systems.

Conclusions:

  • CRISPY-BRED and CRISPY-BRIP offer powerful tools for bacteriophage genome engineering.
  • These methods enhance the potential for phage therapy and detailed genetic dissection of phages.
  • The described approach is adaptable for engineering phages infecting a wide range of bacterial hosts.