Utilizing Golden Gate Assembly to Streamline CRISPR-Cas/NgTET-Based Phage Mutagenesis

Area of Science:

• Molecular Biology
• Synthetic Biology
• Biotechnology

Background:

• Phage engineering holds significant promise for medical and biotechnological applications.
• Customizing phages requires targeted mutagenesis tools for creating designer phages.
• CRISPR-Cas systems are effective for mutagenesis but limited in phage applications due to DNA modifications.

Purpose of the Study:

• To overcome limitations of CRISPR-Cas for phage mutagenesis.
• To develop a novel approach for efficient and targeted phage genome engineering.
• To enable the creation of customized phages with desired traits.

Main Methods:

• Developed a novel approach combining eukaryotic ten-eleven translocation (TET) dioxygenase with CRISPR-Cas.
• Utilized TET enzymes for temporal removal of cytosine modifications on phage DNA.
• Employed Golden Gate cloning for efficient assembly of a vector containing TET and donor DNA for scarless mutagenesis.

Main Results:

• Successfully enabled effective CRISPR-Cas targeting by temporarily removing phage DNA cytosine modifications.
• Facilitated efficient DNA cleavage by Cas enzymes and subsequent mutagenesis.
• Demonstrated a streamlined method for generating customized phage genomes.

Conclusions:

• The developed TET-CRISPR-Cas system significantly advances phage engineering capabilities.
• This approach allows for the efficient generation of designer phages for specific applications.
• The method overcomes previous limitations in phage DNA modification for targeted mutagenesis.